Therapeutic monoclonal antibodies used in Chronic lymphocytic leukemia (CLL) act through complement-mediated cytotoxicity and other mechanisms, and thus depend on the complement (C) availability and activity. Recently we showed abnormal Western analysis pattern of the C5 component in some CLL patients, that was associated with high basal levels of C activation markers, and decreased activity of the classical pathway (CP). We hypothesized that the CP may be constantly activated, and thus exhausted, in a sub-group of CLL patients, identified by the abnormal appearance of C5.
The aim was to study the circulating abnormal C5 and its association with the observed decrease in CP activity, and to search for potential CP-activating factors in CLL plasma.
Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). Activities of the CP and of the CP C5-convertase were studied. C5, C5a and C5b were studied by Western blot analysis. The presence of high molecular weight (HMW) protein complexes, such as IgM or hexameric-IgG, as potential CP activators was assessed by sera fractionation (gel-filtration chromatography) followed by C activation with the obtained HMW, and low molecular weight (LMW) fractions. The levels of IgM were measured. Data from patients with abnormal C5 was compared to normal NC subjects and to patients with normal C5.
In patients with abnormal C5, activities of the CP and of the CP C5-convertase were decreased, compared to NC subjects and to patients with normal C5. The abnormal C5 was identified as a complex of immunoglobulin with C5a (Ig-C5a). In agreement, the high levels of C activation markers showed negative and significant correlation with activity of the CP, but not with the alternative pathway. The data on C activation by the HMW fraction indicated the presence of IgG-aggregates, and not IgM, as the C activating factor in sera.
In some CLL patients, factors such as IgG-aggregates that are present in sera constantly trigger the activation of the CP. This constant activation results in the release of activation products, such as C5a, that form the complex Ig-C5a. Another result is the exhaustion of the CP and the inability to exert CP activity that is comparable to normal subjects. The data provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display Ig-C5a and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.