Analysis of CD16+CD56dim NK cells from CLL patients: evidence supporting a therapeutic strategy with optimized anti-CD20 monoclonal antibodies.

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FcγRIIIA) is well preserved in CD16(+)CD56(dim) cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FcγRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FcγR engagement in CLL patients.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

Circulating von Willebrand factor antigen II in atherosclerosis: a comparison with von Willebrand factor and soluble thrombomodulin.

von Willebrand factor antigen II (vWFAgII) is the 100 kDa propolypeptide of endothelial cell marker von Willebrand factor (vWF). Our aim was to determine the relationship between vWFAgII and mature vWF and an additional endothelial cell marker, soluble thrombomodulin, in atherosclerosis. To do this, we measured levels of all three by enzyme-linked immunosorbent assay in plasma obtained from 24 patients with peripheral vascular disease (PVD), from 25 patients who survived a myocardial infarction [i.e. had ischaemic heart disease (IHD)], and from 47 age- and sex-matched controls. We found raised levels of vWFAgII in PVD (57.3 +/- 15.3 microg/dl; mean +/- standard deviation) and in IHD (53.4 +/- 19.2 microg/dl) compared with the controls (35.7 +/- 12.0 microg/dl; analysis of variance P < 0.001). Raised levels of vWf were found in both groups of patients but raised soluble thrombomodulin was found only in patients with PVD. Levels of vWFAgII correlated with those of vWf (r = 0.45, P < 0.001) but not with soluble thrombomodulin (r = 0.14, P = 0.17), nor any of the major risk factors for atherosclerosis. Our brief study reports raised levels of vWFAgII in atherosclerosis. This may, like that of vWf, be related to endothelial cell damage, although the incomplete correlation between the two implies different metabolic and/or release mechanisms.

A resolution of reported discrepancies in the characteristics of platelet glycoproteins IV (GPIV) and IIIb (GPIIIb).

The terms glycoprotein IV (GPIV) and glycoprotein IIIb (GPIIIb) have been used interchangeably and reports in the literature have indicated this glycoprotein as having a molecular weight variously described as either 88,000 or 97,000, a fast anodal mobility on crossed electrophoresis and either 13 or less than 1 methionine residues on amino acid analysis of the purified glycoprotein. To resolve these discrepancies, we have evaluated the characteristics of GPIV both in whole platelets and after isolation. These studies have shown that the term GPIV defines a protease-resistant platelet surface glycoprotein with Mr 88,330 +/- 2,240 which is immunologically identical with the CD36 differentiation antigen, which migrates with a relatively slow anodal mobility on crossed immunoelectrophoresis and which contains approximately 13 methionine residues per mole.

In vitro and in vivo evaluation of a factor VIII concentrate heat-treated to inactivate HTLV-III/LAV viruses. Favourable effects of heating on the von Willebrand factor.

We report here the results of our evaluation of the effects of a dry heat treatment (96 h at 68 degrees C) to eliminate LAV/HTLV-III virus on factor VIII (FVIII) and von Willebrand factor (vWf) present in an intermediate-purity concentrate. This thermal inactivation appears to have little effect on FVIII. There is an acceptable loss (12.3 +/- 3.6%; n = 25) in FVIII coagulant activity (FVIII: C) and a good in vivo performance in haemophilia A patients. A precise analysis of vWf indicates that whereas the vWf antigen and its ristocetin cofactor activity decrease during heating, there is an increase in potentially functional forms of vWf. Heat treatment induces an increase in high molecular weight forms of vWf and an enhancement in platelet adhesion to collagen. These changes probably explain the correcting effect on the bleeding time of the heated FVIII concentrate in patients with von Willebrand's disease. Thus, this heat-treated concentrate appears to be equivalent to the untreated product in haemophilia A, with the additional benefit of being efficient for the treatment of von Willebrand's disease.