Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody

<div><p>Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested <i>ex vivo</i> in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.</p></div

Patient treatment history.

<p>Patient treatment history.</p

The effect of anti-CD59 antibody on CDC of RTX/CFH MAb nonresponder B-CLL cells with high mean CD59 expression levels (Patient #6).

<p>CDC reactions were carried out with NHS as a complement source. For each condition, the mean percent CDC was divided by the mean percent CDC of the αCon antibody to obtain "fold cytotoxicity". The CDC assay for the α-CFH + RTX condition was performed on a different day from the other conditions.</p

B cell CDC response to mAbs, serum complement activity, CD20 level, and CD59 level for B-CLL patients.

<p>B cell CDC response to mAbs, serum complement activity, CD20 level, and CD59 level for B-CLL patients.</p

Antibody-induced CDC of B-CLL cells from five CLL patients in the presence of PS or NHS.

<p>Panels show B-CLL cell cytotoxicity with the autologous patient serum (PS) from patients 1–5, respectively, in comparison with cytotoxicity using NHS. Cytotoxicity was induced in the presence of RTX plus the addition of either CFH mAb7968 (α-CFH) or isotype-matched negative control antibody 7B2 (α-Con). For each patient and serum type, the mean percent CDC in the presence of the αCFH antibody was divided by the mean percent CDC in the presence of the αCon antibody to obtain "fold cytotoxicity". The p values of the difference between response in PS vs. NHS are shown on the figure. The p values for fold cytotoxicity as a result of α-CFH addition compared to α-Con addition were 0.00017, 0.00060, n.s., n.s., n.s in PS, and 0.00016, 0.00021, 0.000012, 0.00001, and 0.00025 in NHS, for patients 1–5 respectively (n.s = not significant).</p

CDC of B-CLL cells.

<p>(A) CDC of B-CLL cells in the presence and absence of rituximab (RTX). B-CLL cells from each of 11 CLL patients were treated with RTX or left untreated, with NHS added as a source of complement. Mean percent CDC in the presence of RTX was divided by the mean percent CDC in the absence of RTX to obtain "fold cytotoxicity" for each patient. (B) CDC of B-CLL cells from patient 11 treated with a CFH mAb or control mAb in the presence or absence of RTX. B-CLL cells from this patient were treated with CFH mAb7968 or isotype-matched negative control antibody 7B2 with or without RTX, with NHS added as a source of complement. Mean percent CDC was divided by the mean percent CDC of the “no antibody” control to obtain "fold cytotoxicity" for each condition.</p

Patient demographics, B-CLL status, and B cell characterization.

<p>Patient demographics, B-CLL status, and B cell characterization.</p