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. 2011 Jan 24;8(2):157–163. doi: 10.1038/cmi.2010.35

Application of a novel inhibitor of human CD59 for the enhancement of complement-dependent cytolysis on cancer cells

Tao You 1,2,3, Weiguo Hu 2,3, Xiaowen Ge 2, Jingnan Shen 1, Xuebin Qin 2,3
PMCID: PMC4003131  PMID: 21258360

Abstract

Many monoclonal antibodies (mAbs) have been extensively used in the clinic, such as rituximab to treat lymphoma. However, resistance and non-responsiveness to mAb treatment have been challenging for this line of therapy. Complement is one of the main mediators of antibody-based cancer therapy via the complement-dependent cytolysis (CDC) effect. CD59 plays a critical role in resistance to mAbs through the CDC effect. In this paper, we attempted to investigate whether the novel CD59 inhibitor, recombinant ILYd4, was effective in enhancing the rituximab-mediated CDC effect on rituximab-sensitive RL-7 lymphoma cells and rituximab-induced resistant RR51.2 cells. Meanwhile, the CDC effects, which were mediated by rituximab and anti-CD24 mAb, on the refractory multiple myeloma (MM) cell line ARH-77 and the solid tumor osteosarcoma cell line Saos-2, were respectively investigated. We found that rILYd4 rendered the refractory cells sensitive to the mAb-mediated CDC effect and that rILYd4 exhibited a synergistic effect with the mAb that resulted in tumor cells lysis. This effect on tumor cell lysis was apparent on both hematological tumors and solid tumors. Therefore, rILYd4 may serve as an adjuvant for mAb mediated-tumor immunotherapy.

Keywords: CD59, complement, lymphoma, multiple myeloma, rituximab

Introduction

Monoclonal antibody (mAb) therapy is becoming a powerful approach and is increasingly used in the clinic for the treatment of different types of cancer through targeting specific cancer antigens. Rituximab, the first mAb approved for cancer therapy by the Food and Drug Administration, has been successfully used in the treatment of B-cell non-Hodgkin's B-lymphoma (NHL) via specifically targeting the CD20 molecule on the B lymphocyte membrane. Treatment with rituximab has lead to greatly improved clinical outcomes.1 Trastuzumab (Herceptin) is used to treat HER2-positive breast cancer. Other new mAbs are actively being developed. Once the antibodies (Abs) are given, they can then recruit other parts of the immune system to destroy the cancer cells or to enhance the immune response against the cancer. The mechanism of action of these Abs and the host and cellular factors that influence the immune response following Ab treatment are not completely known. The induction of apoptosis, Ab-dependent cell cytotoxicity and complement-mediated cell death (CDC) are the proposed mechanisms of action of these Abs.2

Complement is one of the main mediators of Ab-based cancer therapy via the CDC effect. When cancer therapeutic Abs activate the so-called classical complement pathway, they trigger the formation of the membrane attack complex on cancer cells, leading to the killing of cancer cells through CDC.3 CD59, a critical membrane complement regulator, inhibits membrane attack complex formation by binding to the 8a and 9 (C8a and C9) complement proteins.4, 5, 6 CD59 is universally expressed in normal cells and highly expressed in many kinds of cancer cells, including NHL and chronic lymphocytic leukemia (CLL).3 Extensive clinical and experimental evidence indicates that CD59 is highly effective at protecting NHL and CLL cells from Ab (rituximab or ofatumumab)-mediated CDC.7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19

Many in vivo and in vitro studies indicate that CDC plays a critical role and also interacts synergistically with Ab-dependent cell cytotoxicity in rituximab therapy.3 To further enhance CDC activity, the human IgG1 anti-CD20 mAb ofatumumab has been developed as another new mAb therapeutic.20 Ofatumumab has shown better activity for the treatment of relapsed CLL compared to the activity associated with rituximab.21, 22 Despite these advances, ∼50% of NHL patients are unresponsive to rituximab,23 and some of the responsive patients develop resistance to further rituximab treatment.24 Furthermore, CLL remains incurable with these therapies. Patients undergoing treatment inevitably relapse, become increasingly refractory to treatment, and often acquire high-risk chromosomal abnormalities.22 Thus, there remains a need for more effective therapies in both the upfront and the relapsed settings. Since upregulation of human CD59 (hCD59) is an important determinant of sensitivity to Ab (rituximab and ofatumumab) treatment for NHL and CLL,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 it is imperative for us to develop a molecule capable of abrogating CD59 function in cancer cells and facilitating Ab-mediated cancer therapy. However, anti-hCD59 Abs have demonstrated unacceptable side effects and candidate competitive inhibitor peptides derived from C8 and C9 have also been ineffective.17, 18, 25, 26, 27, 28, 29

Recently, we developed a novel, potent, non-toxic and specific anti-hCD59 inhibitor: the domain 4 of intermedilysin (ILY). This inhibitor is defined as rILYd4.5 Intact ILY only lyses human cells and functions through the formation of large-diameter (250–300 Å) irreversible transmembrane pores. The receptor for ILY is specific for hCD59.30, 31 Using the generation of different mutant ILYs, LaChapelle et al. recently demonstrated that the ILY-hCD59 interaction during the assembly of the pore complex increases host cell susceptibility to the CDC effect.32 Domains 1–3 of ILY are responsible for pore formation, while domain 4 binds to amino acids 42–58 in hCD59. Domain 4 also participates in the binding of hCD59 to C8a and C9.30, 33 We have consistently demonstrated that rILYd4 specifically abrogates hCD59 function in both normal human cells and B lymphoma cells. Furthermore, we demonstrated that rILYd4 (IC50=33 nM) restores the sensitivity of a rituximab-resistant NHL cell line to the rituximab CDC effect. This restoration of sensitivity occurs without off-target toxicity effects on non-target normal cells in vivo (unpublished results). The specificity of this approach originates from the specific cancer Ab. This result indicates that rILYd4 may represent a novel therapeutic approach for the enhancement of Ab-based cancer therapy and that this therapeutic approach may provide a platform for the further development of anti-hCD59 inhibitors. Here, we further investigate whether the rILYd4 enhances the CDC effect on other cancer cells, such as the multiple myeloma (MM) cell line ARH-77 and the human osteosarcoma cell line Saos-2.

Material and methods

Preparation of the rILYd4 and rILY3 proteins and cell lines

The rILYd4 protein was generated by methods previously described.5, 31 The rILY3 fragment contains a portion of domain 4 from ILY: RNIRVKVLGATGLAWEPWRLIYSKNDLPLVPQRNISTWGTTLHPQFEDKVVKDNTD. The RL-7 and ARH-77 cell lines were purchased from American Type Culture Collection. RL-7 and ARH-77 were cultured in RPMI-1640 (Roswell Park Memorial Institute) with 10% fetal bovine serum, 100 IU/ml of penicillin and 100 µg/ml of streptomycin, and the cell lines were maintained in an environment of humidified air with 5% CO2. The Saos-2 cells were cultured in McCoy's 5-A medium with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin, and the cell lines were maintained in an environment of humidified air with 5% CO2. In order to generate rituximab-resistant Ramos cells that were resistant to the rituximab-mediated CDC effect induced with 51.2 µg/ml of rituximab (called as RR51.2), we followed previously reported procedures.15 Briefly, approximately 5×106 cells/ml were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml of penicillin and 100 µg/ml of streptomycin. These cells were further treated for 1 h at 37 °C with 0.2 µg/ml of rituximab (Biogen Idec, Cambridge, CA, USA) and 10% normal human serum (NHS) (Valley Biomedical, Winchester, VA, USA), which was used as a source of complement. The complement-treated cells were washed twice with phosphate-buffered saline (PBS), and the cells were recultured in fresh medium. This process was repeated three times. Following the final culture process, the cells were regarded as 0.2 µg/ml rituximab-resistant cells (RR0.2). The same procedure was repeated to generate other resistant cells by gradually increasing the concentrations of rituximab (0.2, 0.8, 3.2, 12.8 and 51.2 µg/ml). The cells that were resistant to the CDC effect mediated by 51.2 µg/ml of rituximab were termed RR51.2, and these cells were used to perform the experiments.

Fluorescence-activated cell sorting (FACS) analysis

Mouse anti-human CD55 and CD46 mAbs (Serotec, Raleigh, NC, USA), mouse anti-human CD20 mAb (Abcam, Cambridge, MA, USA) and mouse anti-human CD24 and CD59 Abs (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used for FACS analysis. The cell density of each sample was set at 5×106 cells/ml, and the cells were harvested and washed twice with PBS. The cells were suspended in 3% bovine serum albumin/PBS, and the cells were incubated with primary Abs against hCD59, CD55, CD46, CD20 or CD24 (1∶100). The samples were diluted at room temperature for 30 min, washed and then incubated with secondary Ab (goat anti-mouse IgG that was conjugated with FITC) at room temperature for another 30 min. The cells were washed in PBS before analyzing the fluorescence intensity using a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA).

Complement-mediated lysis of human erythrocytes

We assessed the sensitivity of human erythrocytes to human complement-mediated lysis in the presence of rILYd4 and rILY3 by the anti-human erythrocyte Ab-sensitized erythrocyte method, as previously described.31 We used human serum (25% v/v diluted in GVB++ (CompTech, Tylor, TX, USA) as the source of complement. Hemoglobin was measured in the supernatant of lysed erythrocytes by absorbance at 414 nm, and the percent lysis was calculated as follows: % lysis=(test OD414−blank OD414)/(total lysis OD414−blank OD414)×100%.

Complement-dependent cytotoxicity effect

We used the alamarBlue assay to determine cell viability.14 The cells were seeded on a 96-well plate with 5×104 cells per well. Each concentration of rILYd4, rituximab and 10% of NHS was added to a total volume of 200 µl/well and incubated at 37 °C. After 4 h of incubation, 30 µl of alamarBlue and 70 µl of culture medium were added to each well and the preparation was cultured overnight. Cell lysis was assessed by reading the fluorescence using an F-2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) (excitation: 560 nm; emission: 590 nm). The effects of rituximab alone, rILYd4 alone and heat-inactivated NHS were also determined. The percent lysis of each well was calculated as (the reading from the well without any treatment–the reading from the well being tested)/the reading from the well without any treatment Triton×100%.

Statistical analysis

The data were expressed as the mean±SEM. Comparisons of group data were performed using a Student's t-test. The liner correlation between the IC50 of rILYd4 and the CD59 level was analyzed with SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). A P value of <0.05 was considered significant.

Results

rILYd4 enhances the rituximab-mediated CDC effect on lymphoma cell lines

We and others have previously reported that rILYd4 specifically abrogates hCD59 function and sensitizes human erythrocytes to complement-dependent hemolysis.5, 34 We have also demonstrated that rILYd4 inhibits hCD59 activity on the envelope of the HIV-1 virion and sensitizes the HIV-1 virion to CDC.5 Here, we generated rILY3, a smaller fragment of rILYd4. We demonstrated that rILYd4 enhanced complement-dependent hemolysis using a complement-dependent hemolysis assay. In contrast, the treatment of rILY3 did not influence hCD59 activity on human erythrocytes when compared to PBS treatment alone (Supplementary Figure 1). This result, together with the previously reported findings that rILYd4 can specifically compete with anti-hCD59 Abs and intact ILY for binding to hCD59,5 further confirms the specificity of rILYd4 for the abrogation of hCD59 activity.

To further evaluate whether the novel hCD59 inhibitor, rILYd4, enhanced the therapeutic Ab (rituximab)-mediated CDC effects on lymphoma cells in vitro, we used the human follicular lymphoma cell line RL-7 and the rituximab-induced resistant cell line RR51.2. The RR51.2 cells were generated from Ramos cells, which are a Human Burkitt's lymphoma cell line. The rituximab-induced resistant RR51.2 cell line was generated following a previously published protocol.15 Both cell lines, RL-7 and RR51.2, express hCD59 and CD20 on the cell surface, which was determined by FACS analysis (Figure 1a and b). The RR51.2 cells were produced by repeated exposure to increasing concentrations of rituximab, and these cells exhibited resistance to the rituximab-mediated CDC effects. RR51.2 cells were treated with 10fold higher amounts of rituximab compared to the amounts that were sufficient to mediate complement activation in RL-7 cells. Compared to rituximab alone, rILYd4 significantly enhanced either the rituximab (1 µg/ml)-mediated CDC effect on RL-7 cells (IC50: 47 nM) or the rituximab (10 µg/ml)-mediated CDC effect on RR51.2 (IC50: 13.44 nM) cells in a dose-dependent manner (Figure 1c and d). Specifically, the percentage of enhanced cytolysis in the RL-7 cells in the presence of 376 nM of rILYd4 and the RR51.2 cells in the presence of 320 nM of rILYd4 were approximately 35 and 50%, respectively (Figure 1c and d). These results indicate that rILYd4 sensitizes the lymphoma cells to the rituximab-mediated CDC effects (Figure 1c and d). To test whether the abrogation of hCD59 activity by rILYd4 in these lymphoma cells enhances the anticancer activity of rituximab in vitro, we preincubated RL-7 and RR51.2 cells with 376 and 320 nM of rILYd4 to almost completely neutralize the CD59 on the surface of the respective cells. This neutralization was followed by complement activation that was mediated by the different concentrations of rituximab. These rILYd4 doses were selected because they are the minimal dose necessary to achieve an almost maximal anti-hCD59 activity in each cell type (Figure 1c and d). We demonstrated that the pre-incubation of rILYd4 significantly enhanced the CDC effects mediated by the different concentrations of rituximab on both of the cell lines compared to the effects observed following preincubation with PBS (Figure 1e and f). In the presence of rILYd4, we utilized heat-inactivated serum as a source of complement. The heat-inactivated serum did not mediate any significant rituximab-mediated CDC effects on either RL-7 or RR51.2 cells (Supplementary Figure 2). This result directly indicates that the rILYd4-enhanced cancer cell killing effect, which is mediated by rituximab, is complement-dependent. Together, these results document that rILYd4 increases the anticancer activity of rituximab, and that rILYd4 may be a novel approach for use as an adjuvant for rituximab-mediated immunotherapy.

Figure 1.

Figure 1

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rILYd4 sensitized the original RL-7 lymphoma cells and rituximab-induced resistant RR51.2 cells to the rituximab-mediated CDC effect. (a, b) RL-7 cells and RR51.2 cells expressed CD20 and CD59 on the cell surface. Shaded histogram: isotype-matched Abs+FITC secondary Ab staining (negative control); open histogram: anti-hCD59 Ab or anti-CD20 Ab (0.2 µg/ml)+FITC secondary Ab. (c, d) rILYd4 dose dependently enhanced the rituximab (1 or 10 µg/ml)-mediated CDC effect on RL-7 lymphoma cells or RR51.2 cells. Triton X-100 was utilized as a positive control, and all treated cells were 100% cytolysis-positive. Statistical significance is indicated by an asterisk (P<0.01 versus the group treated with rituximab alone). (e, f) Cells were treated with either 376 or 320 nM of rILYd4 to neutralize the hCD59 on lymphoma RL-7 and RR51.2 cells. Statistical significance is indicated by an asterisk (P<0.01 for the rILYd4 group versus the PBS group). Results are expressed as mean±SEM, and the results are derived from three independent experiments. Ab, antibody; CDC, complement-dependent cytolysis; MFI, mean fluorescence intensity; NHS, normal human serum; PBS, phosphate-buffered saline.

rILYd4 enhances the rituximab-mediated CDC effect on a human MM cell line

Although anti-CD20 strategies are very effective in the treatment of various B-cell lymphoproliferative disorders, their therapeutic effects in MM cell lines are undetermined and controversial.35 Recent studies are indicative of the existence of clonogenic CD20-positive precursor B cells in myeloma; thus there is increasing interest in testing anti-CD20 therapies in the disease.35 In order to test whether rILYd4 enhances the rituximab-mediated CDC effect on MM cells, we used FACS analysis to detect CD20-positive and CD59-postive ARH-77 cells and human MM cells (Figure 2a). We demonstrated that rILYd4 sensitized ARH-77 cells to the CDC effect mediated by rituximab (20 µg/ml) in a dose-dependent manner (Figure 2b). Of note, when the cells were treated with different concentrations of rituximab in the absence of rILYd4 (Figure 2c), the rituximab dose-dependent CDC effects on the ARH-77 cells were not as strong as the effects on the RL-7 and RR51.2 cells (Figure 1c and d). This result indicates that the ARH-77 cells were more resistant to the rituximab-mediated CDC effect compared to the other two lymphoma cell lines. Nevertheless, preincubation of the ARH-77 cells with rILYd4 (520 nM) significantly increased the CDC effect that was mediated by all of the different concentrations of rituximab compared to cells that were not pre-treated with rILYd4 (Figure 2c). These results indicate that abrogation of hCD59 activity in CD20-positive MM cells with rILYd4 enhances the rituximab-mediated anticancer killing activity for MM cells.

Figure 2.

Figure 2

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rILYd4 senstized MM ARH-77 cells to rituximab-mediated CDC effect. (a) CD20 and CD59 are expressed on the surface of MM ARH-77 cells. Shaded histogram: isotype-matched Abs+FITC secondary Ab staining (negative control); open histogram: anti-hCD59 Ab or anti-CD20 Ab (0.2 µg/ml)+FITC secondary Ab. (b) rILYd4 dose dependently enhanced the rituximab (20 µg/ml)-mediated CDC effect in the ARH-77 cells. Triton X-100 was utilized as a positive control, and all treated cells were 100% cytolysis positive. Statistical significance is indicated by an asterisk (P<0.01 versus the group treated with rituximab alone). (c) rILYd4 (520 nM) sensitized the MM ARH-77 cells to the rituximab-mediated CDC effect. Statistical significance is indicated by an asterisk (P<0.05 for the rILYd4 group versus the PBS group). Results are expressed as mean±SEM, and the results are derived from three independent experiments. Ab, antibody; CDC, complement-dependent cytolysis; MM, multiple myeloma; NHS, normal human serum; PBS, phosphate-buffered saline.

The degree of ARH-77 resistance to the rituximab-mediated CDC effect also correlates with the expression levels of membrane complement regulatory proteins

We document that ARH-77 cells are very resistant to the rituximab-mediated CDC effect, consistent with the results reported previously by Treon et al.36 It has been demonstrated that the low level of CD20 and high level of CD59 participate in rituximab-mediated resistance in chronic lymphocytic leukemia.36 To investigate the potential mechanism of resistance in ARH77 cells to the rituximab-mediated CDC effect, we also assessed the levels of two other membrane complement regulatory regulators (mCRPs), CD46 and CD55, by FACS analysis (Figure 3a). Of the three different cell lines that were assessed, the ARH-77 cells that had the highest levels of CD20, CD59, CD46 and CD55 were the most resistant to the rituximab-mediated CDC effects in either the presence or the absence of rILYd4 (Figure 3b). This result indicates that CD46 and CD55 may also contribute to the resistance of these cells to the rituximab-mediated CDC effects. In order to investigate the efficacy of rILYd4 for the enhancement of the rituximab-mediated CDC effect on CD20-expressing cells, we further analyzed the IC50 of rILYd4 that is required to kill 50% of the CD20 expressing cells. This included the RL-7, RR51.2 and ARH-77 cell lines, where the effect was mediated by saturated rituximab concentration. We demonstrated that the IC50 of rILYd4 is associated with the CD59 levels in the CD20 expressing cells (Figure 3c). Notably, to compare the efficacy of rILYd4 in enhancing its killing effect on different lymphoma cells and myeloma cells, it is essential to compare other levels of antigen expression, including other mCRPs (CD46 and CD55), on these cells. It is very difficult to obtain cells that express CD20, CD46 and CD55 at the comparable levels necessary to perform these experiments. Nevertheless, we used SPSS 16.0 software (SPSS Inc.) to analyze the liner correlation between the IC50 of the rILYd4 and the CD59 levels, and we demonstrated that the IC50 of rILYd4 positively correlates with the levels of CD59 (Figure 3d). These results further support the notion that rILYd4 abrogates the hCD59 activity and enhances the CDC effect. The efficacy of rILYd4 in the different cells requires further investigation.

Figure 3.

Figure 3

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The resistance of the ARH-77 cells to the rituximab-mediated CDC effect is associated with CD59 and other mCRPs levels. (a) CD46 and CD55 are expressed on the surface of RL-7, RR51.2 and ARH-77 cells. Shaded histogram: isotype-matched Abs+FITC secondary Ab staining (negative control); Open histogram: anti-hCD46 Ab or anti-CD55 Ab (0.2 µg/ml)+FITC secondary Abs. (b) Comparison of CD20 or mCRPs levels on the RL-7, RR51.2 and ARH-77 cells in response to the CDC effect in either the presence or the absence of rILYd4. (c) The dose of rILYd4 required for mediating 50% of the rituximab-mediated cytolysis in RL-7, RR51.2 and ARH-77 cells. Results are expressed as mean±SEM, and the results are derived from three independent experiments. (d) A positive linear correlation was observed between the IC50 of rILYd4 and the expression level of CD59. The positive linear correlation between the IC50 of rILYd4 and the expression level of CD59 was calculated with SPSS 16.0 statistics software. P<0.01 (F=7.803E4). Ab, antibody; CDC, complement-dependent cytolysis; mCRPs, membrane complement regulatory regulators; MFI, mean fluorescence intensity; Rit, rituximab.

rILYd4 enhances the anti-CD24 mAb-mediated CDC effect on the Saos-2 osteosarcoma cell line

In order to further investigate whether the rILYd4 enhances the mAb-mediated CDC effect on solid tumor cells, we selected the Saos-2 osteosarcoma cell line. Over the past 30 years, conventional therapy has failed to improve the survival rate among patients suffering from osteosarcoma.37, 38 However, immunotherapy-based mAbs are a promising new line of therapy that may improve the treatment of patients suffering from this disease. Currently, there are no therapeutic Abs available in the clinic.37 Here, we selected one mouse anti-human monoclonal anti-CD24 Ab, which specifically recognizes the CD24 antigen on osteosarcoma and has been shown to activate the complement system,37 to investigate whether rILYd4 was effective in enhancing the anti-CD24 mediated CDC effect on Saos-2 cell lines. Saos-2 cells, which are a common cell line derived from a human osteosarcoma, express high cell surface levels of CD24 and CD59 (Figure 4a). We demonstrated that either 215 or 430 nM concentration of rILYd4 significantly enhanced the anti-CD24 Ab-mediated CDC effect on Saos-2 cells compared to the untreated group (Figure 4b). This result indicates that rILYd4 may be applicable for the enhancement of Ab-mediated CDC effects on solid tumors, such as osteosarcoma.

Figure 4.

Figure 4

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Recombinant rILYd4 enhanced the anti-CD24 monoclonal Ab (clone SN3)-mediated CDC effect on osteosarcoma Saos-2 cells. (a) CD24 and CD59 are expressed on the surface of Saos-2 cells. Shaded histogram: isotype-matched Ab+FITC secondary Ab staining (negative control); open histogram: anti-hCD59 Ab or anti-CD24 Ab (0.2 µg/ml)+FITC secondary Ab. (b) The Saos-2 cells were effectively sensitized with the 20 µg/ml anti-CD24 monoclonal Ab-mediated CDC effect with 215 and 430 nM of rILYd4. Triton X-100 was utilized as a positive control, and all treated cells were 100% cytolysis-positive. Statistical significance is indicated by an asterisk (P<0.01 versus anti-CD24 Abs alone group). Results are expressed as mean±SEM, and derived from three independent experiments conducted in triplicate. Ab, antibody; MFI, mean fluorescence intensity.

Discussion

Here, we demonstrated that rILYd4, a novel hCD59 inhibitor, enhanced Ab-mediated CDC effects on cells derived from both hematological tumors and solid tumors. These results further support the notion that the recombinant ILYd4 may serve as an adjuvant for mAb-mediated tumor immunotherapy.

Rituximab has been utilized in the clinic for lymphoma immunotherapy for two decades. Rituximab has become the first line drug to treat NHL or CLL alone or in combination with chemotherapy. However, 50% of NHL patients are unresponsive to rituximab or resistant after therapy.25 Extensive evidence indicates that the high expression of mCRPs on the cancer cells influences the efficacy of the rituximab-mediated CDC effect.39 Consistently, we also demonstrated that the high level of CD59 expression was the major cause of lymphoma resistance to rituximab. CD59 mAb may effectively improve the immunotherapeutic result induced by rituximab.39 Meanwhile, in an effort to enhance the efficacy of mAb-induced immunotherapies in other tumor cell lines, a variety of methods, such as Ab, siRNA and peptide treatments, have been used to neutralize or decrease the expression of CD59.40, 41, 42 However, these treatments often produce multiple side effects because they target both tumor cells and normal cells. We selected the novel hCD59 inhibitor rILYd4, which has been shown to have a high affinity to CD595 and has not shown signs of cytoxicity in normal cells (unpublished data), to improve or sensitize the resistant lymphoma cell lines in vitro. There is a positive relationship between the IC50 dose of rILYd4 with CD59 expression in the three different cell lines tested, which confirms that the rituximab-resistant lymphoma cell lines were associated with CD59 expression. Additionally, rILYd4 effectively abrogates CD59 function and facilitates the rituximab-based immunotherapy in the rituximab-resistant lymphoma cells in vitro. This provides a new approach to inhibit the ability of CD59 to ameliorate the rituximab immunotherapeutic effect in resistant lymphoma cells.

MM is a clonal B-cell malignancy characterized by aberrant expression of plasma cells within the bone marrow. In the clinic, researchers have found an intermediate to high level of CD20 expression in 15–20% of MM cells.43, 44 Moreover, studies have also suggested the existence of clonogenic CD20-positive precursor B cells in the disease. Thus, there is increasing interest in testing anti-CD20 therapies in myeloma. However, not all CD20+ patients respond to rituximab; in fact, only 10% of the CD20+ patients are responsive.35 In this paper, we demonstrated that the expression of the complement regulator CD59 is associated with resistance to rituximab-mediated complement lysis in the ARH-77 MM cell line. rILYd4 was effective in abrogating the function of hCD59 in the ARH-77 cell lines. After the inhibition of hCD59 function with rILYd4, the ARH-77 cell lysis of rituximab-mediated complement was increased. rILYd4 sensitized the ARH-77 cells to rituximab. There was a synergistic effect between rituximab and rILYd4 on the MM cell lines, which provides a new prospect for the treatment of MM. However, the abrogation of the hCD59 function may not completely resolve the rituximab resistance in MM. The incomplete resolution may be associated with the high expression levels of CD46 and CD55 in the ARH-77 cells, which also play a role in the CDC effect that is based on the rituximab therapy in MM. This hypothesis requires further investigation.

Osteosarcoma is a type of malignant tumor associated with bone tissue in humans. This type of tumor often occurs during the second decade of life. The 5-year survival rate is below 70%, even following an effective course of neo-adjuvant chemotherapy and regular surgery.45 Immunotherapy is regarded as a very promising method for protection against relapse and metastasis of osteosarcoma, and immunotherapy has been shown to extend the survival period.37 Moreover, high levels of hCD59 expression were discovered in osteosarcoma cell lines.23 This finding suggests that the high levels of hCD59 may be the obstructive factor in osteosarcoma immunotherapy. Here, we utilized the rILYd4 to inhibit hCD59 function in the Saos-2 osteosarcoma cell line. We documented that rILYd4 sensitized Saos-2 cells to the CDC effect induced by anti-CD24 mAb and exhibited a strong synergistic effect with anti-CD24 mAb to treat the Saos-2 osteosarcoma cell line. In summary, rILYd4 may be used in both malignant tumors residing in the hematological system and solid tumors to enhance the CDC effect based on the mAb.

Acknowledgments

This work was supported by the US NIH through grant RO1 AI061174 (XBQ) and grant R21 CA141324 (XBQ), the Harvard Technology Development Accelerator Fund (XBQ), and the Fund of the China Scholarship Council No. 2008638052 (TY). We are grateful to Ms Annie Qin, a Harvard University undergraduate student, for helpful editorial assistance.

Footnotes

Note: Supplementary information is available on the Cellular & Molecular Immunology website(http://www.nature.com/cmi/).

Supplementary Information

Supplementary information
Click here for additional data file. (42.7KB, pdf)

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