Cytokines affect resistance of human renal tumour cells to complement-mediated injury.

Overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells may hamper the effect of immunotherapy with complement-activating monoclonal antibody (MoAb). Therefore, it is important to investigate whether cytokines can downregulate the expression of mCRP on tumour cells. In this study, the effect of 10 cytokines on the expression of the mCRP CD46, CD55 and CD59 and the renal tumour-associated antigen G250/MN/CAIX on four human renal tumour cell lines and proximal tubular epithelial cells was determined by flow cytometry. In addition, it was measured whether changes in the expression of the classical pathway regulatory proteins CD55 and CD59 had an effect on C3 deposition and lysis. Interleukin-1beta (IL-1beta) consistently downregulated the expression of CD46 and CD59; IL-4 consistently downregulated the expression of CD46 and transforming growth factor-beta1, consistently downregulated the expression of both CD46 and CD55. However, treatment with IL-1beta and IL-4 also decreased the expression of G250/MN/CAIX. Changes in the expression of CD55 and CD59 were associated with changes in the amount of C3 deposited and the extent of complement-mediated lysis, respectively. This suggests that clinical immunotherapy, consisting of treatment with cytokines and MoAb, may induce either up- or downregulation of CD55 or CD59 and thus affect the effectiveness of immunotherapy with MoAb.

A possible role of CD46 for the protection in vivo of human renal tumor cells from complement-mediated damage.

It is still unclear which membrane-bound regulatory proteins (mCRP) are important in vivo to protect tumor cells from complement-mediated damage. To address this question, the expression levels of CD46, CD55, and CD59 were measured semi-quantitatively in situ on renal cell carcinomas and compared with the expression level and cellular distribution of these mCRP in proximal tubuli within each patient (n = 31). It was also determined whether the expression of mCRP on tumor cells is associated with deposition of C3d and C5b-9. CD46 expression was decreased on tumor cells; in contrast, CD55 was expressed on tumor cells (12 out of 31 samples), while it was not detected on proximal tubular epithelial cells (PTEC). Also, expression of CD59 on tumor cells was increased as compared with its expression on PTEC. Furthermore, the localization on the cell surface of mCRP as observed on PTEC was altered on tumor cells. Because expression of mCRP may limit a complement-mediated anti-tumor response, we determined whether complement deposition was associated with the expression level of CD46, CD55, and CD59. The presence of C3d on tumor cells was associated with a low expression level of CD46 (p < 0.02). The expression level of CD46 was also associated with a low tumor stage (p < 0.04). The results suggest that in vivo CD46 plays a role in the protection of human renal tumor cells from complement-mediated injury.

A bispecific monoclonal antibody directed against both the membrane-bound complement regulator CD55 and the renal tumor-associated antigen G250 enhances C3 deposition and tumor cell lysis by complement.

Tumor cells may inhibit the induction of a complement-mediated inflammatory response through overexpression of membrane-bound regulators of complement activation. Therefore, it is of interest to determine the most efficient approach to block these membrane-bound complement regulators on tumor cells. In the present study, we first generated a bispecific mAb directed against both CD55, using the functional blocking mAb MBC1, and the highly expressed HLA class I molecule as a model for a tumor-associated Ag, using the mAb W6/32. Tumor cells opsonized with bispecific mAb W6/32*MBC1, then exposed to complement and subsequently stained for C3 deposition, were assessed by flow cytometric analysis. We found that opsonization with W6/32*MBC1 resulted in a 92% enhancement of C3 deposition on renal tumor cells as compared with opsonization with W6/32 alone and a 17% enhancement of the C3 deposition as compared with incubation with a mixture of both parental mAb. Based on these results, we developed a bispecific mAb recognizing both CD55 and the relatively low expressed renal tumor-associated Ag G250. Increasing concentrations of the bispecific mAb G250*MBC1 resulted in a 25 to 400% increase in C3 deposition on renal tumor cells as compared with C3 deposition in the presence of the parental mAb G250 alone. G250*MBC1 enhanced C3 deposition by 21% in comparison with a mixture of both parentals. Furthermore, opsonization of tumor cells with G250*MBC1 rendered these cells more sensitive to complement-mediated lysis. In conclusion, the bispecific mAb G250*MBC1 induces deposition of C3 and tumor cell lysis more efficiently than G250 alone.

Expression of CD46, CD55, and CD59 on renal tumor cell lines and their role in preventing complement-mediated tumor cell lysis.

Nucleated cells are protected from complement-mediated injury by the expression of membrane-bound regulators of complement activation (mRCA) CD46, CD55, and CD59. Increased expression of these mRCA may be a mechanism by which tumor cells protect themselves from complement-mediated injury and prevent an inflammatory response. In the present study, we have investigated whether human renal tumor cell lines and cultured proximal tubular epithelial cells express CD46, CD55, and CD59 and whether these mRCA influence complement-mediated lysis of these cells. The expression of CD46, CD55, and CD59 was measured by flow cytometry. To determine the effect of mRCA on lysis, tumor cells were opsonized with complement activating anti-HLA class l mAb. Lysis was measured in the presence or absence of anti-CD46, anti-CD55 or anti-CD59 mAb and serum as a source of complement, using a 51Cr release assay. Flow cytometric analysis revealed that renal tumor cell lines and proximal tubular epithelial cells all express CD46, CD55, and CD59. Lysis of renal tumor cell lines in the presence of rabbit serum depended on the number of HLA class I molecules expressed by the tumor cells. Using human serum, complement-mediated lysis was decreased by at least one-third as compared with rabbit serum. The susceptibility of renal tumor cells for complement-mediated lysis could be increased up to the level observed with rabbit serum by inhibiting the function of CD59. Inhibition of the function of CD46 or CD55 with mAb directed against these mRCA had no substantial effect on lysis. We conclude from this work that renal tumor cells and proximal tubular epithelial cells express CD46, CD55, and CD59. Of these mRCA, CD59 is most efficient in preventing complement-mediated lysis of these cells. Expression of mRCA on tumor cells may influence the effectiveness of immunotherapy with tumor-associated mAb.