Effectivity Of Complement-Dependent Cytotoxicity Induced By Therapeutic Antibodies In Acute Lymphoblastic Leukemia Is Dictated By Antigen Expression Levels and Curtailed By Membrane-Bound Complement Regulatory Proteins

Membrane-bound complement regulatory proteins (mCRPs) are known to protect cells from bystander lysis and spontaneous activation of complement. In the case of antibody therapy these mCRP’s may down-modulate antibody-induced complement-dependent cytotoxicity (CDC). Two of these mCRPs, CD55 (DAF) and CD59 (MIRL), have been suggested as modulators of CDC. However, no strong correlation between their expression level and target cell lysis has been demonstrated thus far. Our goal was to elucidate the role of these mCRPs in the efficacy of antibody-induced CDC.

We tested the effectivity of CDC induced by the therapeutic antibodies Alemtuzumab (Alt, anti-CD52), Rituximab (Rtx, anti-CD20), and Ofatumumab (Ofa, anti-CD20) in 16 cell lines showing differential expression of target antigens and mCRPs. These cell lines were generated in our laboratory from primary acute lymphoblastic leukemia (ALL) cells and their phenotype and karyotype greatly resembles the primary malignancy. CDC was induced by incubation of target cells with a serial dilution of the respective antibody in the absence or presence of human serum for a period of 18 hours. Subsequent target cell death was determined using flow cytometry by measuring the absolute number of surviving cells (propidium iodide negative, CD19 positive) per sample, using a fixed number of CytoCount™ beads as an internal reference, and calculating the magnitude of cell loss compared to untreated control samples.

A strong correlation was found between antigen expression level and CDC induction by Alt, Rtx, and Ofa. Interestingly, considerable deviation from this correlation was observed especially in cell lines with intermediate antigen expression levels. To test whether this was due to differential expression of mCRPs, surface expression levels of CD55 and CD59 were measured by flowcytometry. The magnitude of CDC strongly correlated to the mCRP expression in the cell lines with intermediate antigen expression (MFI 1128±104 (CD52, n=5) 796±132 (CD20, n=3)), resulting in 51% (Alt) and 75% (Ofa) reduction of antibody-induced lysis in cell lines with high CD55/CD59 expression compared to cell lines with low CD55/CD59 expression. In the cell lines with high antigen expression (MFI 2802±67 (CD52) 2136±692 (CD20)), the protective effect of co-expression of mCRPs appeared to be overpowered by the high target antigen expression levels.

To study the individual contribution of CD55 and CD59 to the protective effect against CDC, we induced the expression of CD55 and CD59 separately in the cohort of target cell lines with intermediate target antigen expression. Cell lines were transduced, using the bicistronic LZRS vector and the ϕ-NX-A retrovirus packaging cell line, with a construct coding for CD55 or CD59, respectively, coupled to the truncated nerve growth factor receptor (tNGFR) via an internal ribosome entry site (IRES)-sequence. Cells positive for membrane expression of the tNGFR marker and CD55 or CD59 were sorted using flowcytometric cell sorting. This resulted in cell lines with significantly increased expression levels of the respective mCRP compared to their MOCK transduced counterparts, yet never exceeding physiological levels as found in the original ALL cell lines. When compared to MOCK transduced cell lines, all CD55 transduced cell lines (n=4) showed a decreased sensitivity for Alt-induced CDC with a maximum decrease of 69%. Similarly, forced induction of CD59 expression significantly decreased the sensitivity to CDC compared to MOCK transduced cell lines when using Alt (maximum decrease 52%), Rtx (maximum decrease 56%), or Ofa (maximum decrease 30%) (n=5).

In conclusion, we show that efficacy of CDC induction by therapeutic antibodies is dictated by the level of target antigen expression, but is dominantly influenced by expression of the mCRPs CD55 and CD59. This restraining effect on sensitivity to antibody-induced CDC was observed using Alemtuzumab as well as Rituximab and Ofatumumab, suggesting a general mechanism independent of target antigen or type of antibody. Therefore, mCRP expression on malignant cells is likely to be an important factor in the sensitivity to antibody treatment, especially when cells with intermediate antigen expression are targeted.