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Clinical Cancer Research Vol. 5, 4259-4263, December 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

The Ch14.18-GM-CSF Fusion Protein Is Effective at Mediating Antibody-dependent Cellular Cytotoxicity and Complement-dependent Cytotoxicity in Vitro1

Ayse Batova, Arvid Kamps, Stephen D. Gillies, Ralph A. Reisfeld and Alice L. Yu2

Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of California, San Diego, San Diego, California 92103-8447 [A. B., A. L. Y.]; Isala Clinics, Weezenlanden Hospital, Department of Pediatrics, 8000 GM Zwolle, the Netherlands [A. K.]; Fuji ImmunoPharmaceuticals Corporation, Lexington, Massachusetts 02173 [S. D. G.], and Department of Immunology, Scripps Research Institute, La Jolla, California 92037 [R. A. R.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Granulocyte/macrophage-colony stimulating factor (GM-CSF) is very effective at enhancing antibody-dependent cellular cytotoxicity (ADCC) mediated by granulocytes and monocytes. Recently, a fusion protein consisting of GM-CSF and chimeric human/mouse anti-ganglioside GD2 antibody Ch14.18 (Ch14.18-GM-CSF) has been generated to improve the effectiveness of immunotherapy by directing GM-CSF to the tumor microenvironment and prolonging its relatively short half-life. In this study, we examined the ability of this fusion protein to enhance the in vitro killing of GD2- expressing human neuroblastoma cells by granulocytes and mononuclear cells, as well as by complement. The Ch14.18-GM-CSF fusion protein was equally effective as the combination of equivalent amounts of free Ch14.18 and GM-CSF in mediating the killing of NMB7 neuroblastoma cells by granulocytes from seven of eight neuroblastoma patients. The fusion protein was also equally effective as the combination of Ch14.18 and GM-CSF in mediating ADCC by neuroblastoma patients’ mononuclear cells. In addition, the fusion protein was as effective as Ch14.18 alone in directing complement-dependent cytotoxicity against NMB7 cells. Our results demonstrate that the biological activities expressed by ADCC and complement-dependent cytotoxicity of both monoclonal antibody Ch14.18 and GM-CSF are retained by the Ch14.18-GM-CSF fusion protein and lend further support for future clinical trials of this fusion protein in patients with neuroblastoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Advances in cancer therapy in the last three decades have transformed the majority of childhood cancers from uniformly fatal diseases to largely curable illnesses. Unfortunately, the outcome of advanced stage neuroblastoma, which comprises more than one-half of all neuroblastoma, remains dismal despite surgery, radiation, intensive chemotherapy, and bone marrow transplantation. Clearly, new therapeutic strategies for the treatment of neuroblastoma are urgently needed. A promising approach is targeted therapy with mAbs3 directed against human tumor-associated antigens. In this regard, immunotherapy with mAbs directed against the neuroblastoma-associated antigen, disialoganglioside GD2, has been actively pursued (1, 2, 3) . The GD2 antigen is ideal for mAb-mediated therapy of neuroblastoma because it is expressed at high density in the vast majority of human neuroblastoma cells but is absent in normal tissues excluding neurons, skin melanocytes, and peripheral pain fibers, where it is poorly expressed (4 , 5) . To minimize immunogenicity, a human-mouse chimeric antibody directed against ganglioside GD2 (Ch14.18) was developed by fusing the cDNA sequences encoding the constant portion of human {gamma}1 heavy chain and {kappa} light chain with those encoding the variable portions of immunoglobulin from the murine hybridoma 14.18 (6) . Ch14.18 was demonstrated to be very effective in mediating ADCC against neuroblastoma cells in the presence of human granulocytes and mononuclear cells (7) , as well as in directing complement against neuroblastoma and melanoma (8) . Phase I clinical trials of Ch14.18, conducted by us and others in children with neuroblastoma, revealed that Ch14.18 had therapeutic efficacy and was fairly well tolerated with proper supportive care (9 , 10) .

Many cytokines enhance effector cell functions, particularly in mediating ADCC (11, 12, 13, 14) . Our recent studies demonstrated that GM-CSF enhances anti-GD2-mediated ADCC by granulocytes of normal individuals as well as those of neuroblastoma patients (15) . These and other encouraging results have prompted the use of GM-CSF, in conjunction with mAbs in clinical trials, for the treatment of colorectal carcinoma (16 , 17) and neuroblastoma (18) . GM-CSF augmented ADCC activity of mononuclear cells and granulocytes against colorectal carcinoma cells (16 , 17) and neuroblastoma (18) , respectively. In both clinical trials, the use of GM-CSF in conjunction with tumor antigen-specific mAbs demonstrated therapeutic efficacy. In view of the effectiveness by which cytokines stimulate the proliferation of various effector cells and enhance their ADCC activities, efforts have been made to target cytokines to the tumor microenvironment by using genetically engineered fusion proteins consisting of cytokines and mAbs directed against tumor-associated antigens. For example, Ch14.18 has been fused to IL-2, GM-CSF, TNF-{alpha}, and TNF-{beta} (19, 20, 21) . These fusion proteins were shown to bind GD2 on neuroblastoma and melanoma cells, and Ch14.18-IL2 was reported to stimulate CD8+ T-cell-mediated killing of these tumors in syngeneic mouse tumor models. Previously, the Ch14.18-GM-CSF fusion protein was found to bind purified GD2 antigen similarly as Ch14.18 but was five times less active than recombinant GM-CSF in stimulating the proliferation of GM-CSF-dependent cells (19) . However, a more important question to answer is whether fusion of GM-CSF to mAb Ch14.18 interferes with the well-known biological activities of Ch14.18 and GM-CSF such as ADCC and CDC.

Here, we investigated for the first time the ability of the Ch14.18-GM-CSF fusion protein to stimulate ADCC by granulocytes and mononuclear cells of neuroblastoma patients and to mediate complement-dependent lysis of human neuroblastoma cells. Our results indicated the Ch14.18-GM-CSF fusion protein to be equally effective as a mixture of equivalent amounts of Ch14.18 and GM-CSF in mediating ADCC by granulocytes and mononuclear cells. This finding indicates that the ability of Ch14.18 to mediate ADCC, and of GM-CSF to enhance ADCC, is preserved in the fusion protein. At concentrations up to 0.2 µg/ml, the Ch14.18-GM-CSF fusion protein was equally effective as mAb Ch14.18 in directing complement-dependent lysis of neuroblastoma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Antibodies.
The neuroblastoma cell line NMB7 was graciously provided by Dr. Shuen-Kuei Liao, Chang Gung Medical College, Tao-Yuan, Taiwan. The human-mouse chimeric anti-GD2 antibody, Ch14.18 (6) , and the Ch14.18-GM-CSF fusion protein (19) were developed and characterized as described previously.

Patient Population.
The patient population consisted of eight high-risk neuroblastoma patients who had recurrent or refractory disease after at least one regimen of multiagent chemotherapy and autologous bone marrow transplant in four or eight patients (Table 1)Citation .


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  		Table 1 Antibody-dependent lytic activity of GM-CSF-stimulated granulocytes and mononuclear cells

 
Isolation of Granulocytes and Mononuclear Cells.
Granulocytes were isolated from eight neuroblastoma patients characterized in Table 1Citation . Briefly, patients’ heparinized whole blood or bone marrow were centrifuged on a Ficoll-Paque density gradient at 400 x g for 30 min. The resulting pellet, consisting of RBCs and granulocytes, was washed with RPMI 1640, resuspended in hemolytic buffer (0.15 M NH4Cl and 0.01 M NaHCO3), and incubated for 2 min at 37°C to lyse RBCs. The remaining granulocytes were washed twice with RPMI 1640 and resuspended in medium supplemented with 10% FBS and 2 mM glutamine (complete media). Mononuclear cells were isolated from the interphase after Ficoll-Paque density centrifugation and then washed twice with RPMI 1640 before being suspended in complete media. Granulocyte and mononuclear cell populations were determined to be >99% pure by differential cell counting after staining cells with Wright’s stain. Cell viability was >99%, as determined by trypan blue exclusion.

ADCC.
The lytic activity of neutrophils and mononuclear cells was determined by a 51Cr-release assay using a neuroblastoma cell line, NMB7, as target cells (1 x 106) that were labeled with 0.2 mCi of Na251CrO4 for 4 h at 37°C. After three washes with RPMI 1640, target cells were resuspended in complete media at 2 x 104 cells/ml, and 100 µl of this cell suspension were added to each well of a round-bottomed, 96-well plate containing either Ch14.18, Ch14.18 and GM-CSF, or Ch14.18-GM-CSF fusion protein and effector cells at E:T ratios of 100, 50, 25, and 10. Ch14.18 and GM-CSF were added to wells at concentrations matching those of the fusion protein on a molar basis. Final Ch14.18 concentrations were 0.05, 0.1, 0.5, and 1.0 µg/ml, and those of GM-CSF were 13, 26, 130, and 260 ng/ml, respectively. All conditions were performed in triplicate, and cells were incubated for 4 or 18 h at 37°C for granulocytes and mononuclear cells, respectively. Target cell lysis was determined by counting 100 µl of supernatant in a gamma scintillation counter after the collection of intact cells by centrifugation. Total and spontaneous lysis were determined by incubation of target cells in 0.2% SDS or in complete medium, respectively. The percentage of lysis was calculated by the formula:

The percentage of lysis is expressed by LU that were determined by using the exponential fit equation described by Pross et al. (22) . One lytic unit is defined as the number of effector cells required to obtain 20% lysis of target cells.

CDC.
NMB-7 cells were labeled with Na251CrO4 as described above for the ADCC assay. Labeled cells were plated at 2000 cells/well in a round-bottomed, 96-well plate containing increasing concentrations of either Ch14.18, Ch14.18 plus GM-CSF, or the Ch14.18-GM-CSF fusion protein and then incubated at 37°C for 1 h. The concentrations of Ch14.18 were matched to those of the fusion protein on a molar basis. Human sera from two normal donors were diluted 2- and 10-fold with RPMI 1640, and 100 µl were added to the wells containing target cells and incubated for 1.5 h at 37°C. Heat-inactivated serum was used as a control to ensure measurement of complement-specific lysis. Specific target cell lysis was determined as described above. Antibody-mediated CDC was determined by subtracting the percentage of tumor cell lysis attributable to complement alone.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ADCC.
The binding of the Ch14.18-GM-CSF fusion protein to the target antigen GD2 was compared with that of Ch14.18 in a previous study (19) . The results of an ELISA assay established that there was no significant difference in the binding of the Ch14.18-GM-CSF fusion protein and Ch14.18 to GD2. To determine whether the immune-modulatory effect of GM-CSF and the biological activities of Ch14.18 are preserved in the Ch14.18-GM-CSF fusion protein, we performed ADCC assays using as effector cells granulocytes isolated from eight neuroblastoma patients and target cells from the neuroblastoma cell line NMB7. In two representative experiments (Fig. 1)Citation , the capacity of the Ch14.18-GM-CSF fusion protein to mediate tumor cell lysis by granulocytes was equivalent to that of the mixture of Ch14.18 and GM-CSF at all concentrations tested. Antibody-dependent tumor cell lysis peaked at 0.5–1 µg/ml of Ch14.18 with a GM-CSF concentration of 130–260 ng/ml. Results of ADCC obtained with granulocytes from eight neuroblastoma patients are summarized in Table 1Citation . The lytic activity mediated by Ch14.18 varied from patient to patient, but the addition of GM-CSF augmented tumor cell lysis mediated by Ch14.18 in six of eight samples, as observed by us previously (7) . Importantly, tumor cell lysis mediated by the Ch14.18-GM-CSF fusion protein was essentially the same as that achieved by equivalent amounts of Ch14.18 plus GM-CSF in all but one patient (P1015). Furthermore, fusion protein-mediated tumor cell lysis was equivalent to that mediated by a mixture of Ch14.18 and GM-CSF, with granulocytes isolated from two different sources from patient U13, i.e., peripheral blood and bone marrow. In addition, ADCC activity of peripheral blood mononuclear cells obtained from the same neuroblastoma patient (U13) on two occasions was the same with the fusion protein or a mixture of mAb Ch14.18 and GM-CSF at equivalent concentrations (Table 1)Citation . However, GM-CSF, either as part of the Ch14.18-GM-CSF fusion protein or as a separate entity, did not augment ADCC of peripheral blood mononuclear cells to the same extent as granulocytes of some patients (Table 1)Citation . Taken together, our results demonstrate that the fusion of GM-CSF to Ch14.18 does not alter the biological function of Ch14.18 in mediating ADCC of granulocytes and mononuclear cells nor affect the ability of GM-CSF to stimulate ADCC activity of these effector cells in vitro.



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  		Fig. 1. Stimulation by GM-CSF of antibody-mediated lysis of neuroblastoma cells by granulocytes. Granulocytes isolated from peripheral blood of neuroblastoma patients were used as effector cells against NMB7 tumor cells in the presence of increasing concentrations of Ch14.18 plus GM-CSF (•) or Ch14.18-GM-CSF fusion protein ({blacktriangleup}) in a 4-h 51Cr-release assay as described in "Materials and Methods." The concentrations of free Ch14.18 and GM-CSF were matched on a molar basis to those present in the fusion protein. , patient 1006; ----, patient U13. At least three independent ADCC experiments were performed for each of these two patients. Lytic activity of granulocytes alone was 0.5 ± 0.5 LU and 0 ± 0 LU for patients U13 and 1006, respectively. Specific lysis of target cells by up to 1109 g/ml of Ch14.18 or Ch14.18-GM-CSF alone was 1.0 ± 1.4%. Bars, SD.

 
CDC.
To determine whether the Ch14.18-GM-CSF fusion protein is able to direct CDC and whether this ability compares with that of mAb Ch14.18 alone, we performed CDC assays using sera from two normal donors as the complement source and cultured NMB7 human neuroblastoma cells as target. The results of a representative experiment among three experiments shown in Fig. 2Citation indicated that complement-dependent lysis was similar when mediated by either the Ch14.18-GM-CSF fusion protein or mAb Ch14.18. Interestingly, however, in two of three experiments, at concentrations of Ch14.18 >0.2 µg/ml, the fusion protein appeared to be somewhat less effective than Ch14.18 alone in directing complement-dependent lysis.



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  		Fig. 2. Complement-dependent lysis of neuroblastoma cells. Serum from a normal donor diluted 2-fold was used as the complement source against NMB7 tumor cells in a 1.5-h 51Cr-release assay after a 1-h preincubation at 37°C of NMB7 cells with increasing concentrations of Ch14.18 alone (•) or the Ch14.18-GM-CSF fusion protein ({blacktriangleup}). The CDC assay was performed three times using sera from two different donors. Total lysis, 846 ± 113 cpm; spontaneous lysis, 75 ± 17 cpm; lysis by human complement alone, 5 ± 8 cpm. Bars, SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Ch14.18-GM-CSF fusion protein was reported previously to bind GD2 to a similar extent as mAb Ch14.18 (19) . We now demonstrate for the first time that this same fusion protein is capable of mediating ADCC against human neuroblastoma cells by granulocytes and mononuclear cells of neuroblastoma patients. Furthermore, the Ch14.18 moiety of the Ch14.18-GM-CSF fusion protein was equally effective as equivalent amounts of mAb Ch14.18 in mediating ADCC, and the GM-CSF moiety of this fusion protein was as effective as GM-CSF in enhancing ADCC mediated by granulocytes and mononuclear cells, when the mAb Ch14.18 and GM-CSF concentrations were matched on a molar basis. Previous in vitro studies with Ch14.18-IL-2 fusion protein (21) or a mAb-TNF-{alpha} conjugate (23) demonstrated that these were more effective in mediating ADCC compared with cytokine alone (21 , 23) or mAb alone (21) . A more meaningful comparison of ADCC activities is to compare those achieved with either the fusion protein or the combination of mAb and cytokine at equivalent concentrations. As observed previously by us (7 , 15 , 18) , GM-CSF generally enhanced Ch14.18-mediated ADCC by granulocytes; however, now we demonstrated that this activity of GM-CSF was not diminished when fused to Ch14.18. The Ch14.18-GM-CSF fusion protein performed as well as Ch14.18 alone in directing CDC. However, at concentrations of Ch14.18 generally >0.2 µg/ml, the fusion protein appeared to be somewhat less effective in directing CDC than free Ch14.18. It is possible that at high concentrations, the structural features of the fusion protein may impose some steric hindrance to complement fixation in vitro.

Functions of immune effector cells are usually suppressed in most cancer patients, and many chemotherapeutic drugs induce immunosuppression and neutropenia (24 , 25) . Consistent with this observation, we found that granulocytes from six of eight neuroblastoma patients mediated very low ADCC with mAb Ch14.18 alone, yet in all but one case, GM-CSF enhanced granulocyte and mononuclear cell ADCC. The ability of GM-CSF to increase the production of granulocytes and mononuclear cells as well as to enhance their cytotoxic activities against tumor cells is well documented (11, 12, 13, 14, 15, 16) . In addition, GM-CSF can also affect the migration of granulocytes (7 , 26) , resulting in their increased accumulation at tumor sites (27) . In view of the effects of GM-CSF on these effector cells, particularly granulocytes, the use of a Ch14.18-GM-CSF fusion protein in the treatment of neuroblastoma would be of considerable interest, especially because our present study indicates that the Ch14.18-GM-CSF fusion protein is equally effective in mediating ADCC in vitro as are mixtures of Ch14.18 and GM-CSF at equivalent concentrations. More importantly, on the basis of our in vivo studies with Ch14.18-IL-2 (28 , 29) , the Ch14.18-GM-CSF fusion protein would be expected to target GM-CSF and thereby direct granulocytes and mononuclear cells to the tumor microenvironment far more effectively than a combination of Ch14.18 and GM-CSF. In this regard, in addition to studies with Ch14.18-IL-2, earlier studies demonstrated that mAb-cytokine conjugates administered to mice could target cytokines to tumor sites more effectively than the administration of free cytokine (30) . Furthermore, the fusion of GM-CSF to Ch14.18 would not only increase the half-life of GM-CSF but would also avoid or minimize toxicities of GM-CSF, as was observed with a bispecific antibody targeting TNF-{alpha} to tumor sites (31) . A HAMA or an anti-GM-CSF response may occur against the Ch14.18-GM-CSF fusion protein. However, on the basis of results of in vivo studies (27 , 28) obtained with a similar fusion protein, Ch14.18-IL-2, it does not appear that a HAMA or anti-GM-CSF response would occur or occur to any significant level to affect the antitumor effect of the Ch14.18-GM-CSF fusion protein. The results of the studies on the Ch14.18-IL-2 fusion protein demonstrated that the fusion protein was much more effective than the mixture of Ch14.18 and IL-2 in suppressing the growth of disseminated metastases of neuroblastoma to bone marrow and liver. Furthermore, the occurrence of a HAMA response may actually be beneficial because of the generation of an anti-id response. Recent studies have shown that generating an anti-id response was effective in obtaining an antitumor response (32 , 33) .Taken together, our results lend strong support for conducting clinical trials with a Ch14.18-GM-CSF fusion protein in neuroblastoma patients.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants FD-R-00952 and 95-5470 from the FDA (to A.L.Y.), Grant CA 42508-13 from the NIH (to R.A.R.), and in part by Cindy Matters Funds and Grant MOI RR00827 from the general Clinical Research Center program of the National Center for Research Resources, NIH. Back

2 To whom requests for reprints should be addressed, at Division of Pediatric Hematology/Oncology, UCSD Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8447. Back

3 The abbreviations used are: mAb, monoclonal antibody; ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; GM-CSF, granulocyte/macrophage-colony stimulating factor; IL-2, interleukin 2; TNF, tumor necrosis factor; LU, lytic unit(s); HAMA, human antimouse antibody. Back

Received 4/29/99; revised 9/ 9/99; accepted 9/21/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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C. Schwegler, A. Dorn-Beineke, S. Nittka, C. Stocking, and M. Neumaier
Monoclonal Anti-idiotype Antibody 6G6.C4 Fused to GM-CSF Is Capable of Breaking Tolerance to Carcinoembryonic Antigen (CEA) in CEA-Transgenic Mice
Cancer Res., March 1, 2005; 65(5): 1925 - 1933.
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U. Pertl, H. Wodrich, J. M. Ruehlmann, S. D. Gillies, H. N. Lode, and R. A. Reisfeld
Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma
Blood, January 15, 2003; 101(2): 649 - 654.
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L. S. Metelitsa, S. D. Gillies, M. Super, H. Shimada, C. P. Reynolds, and R. C. Seeger
Antidisialoganglioside/granulocyte macrophage-colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on Fcgamma RII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis
Blood, May 13, 2002; 99(11): 4166 - 4173.
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C.-L. Tso, A. Zisman, A. Pantuck, R. Calilliw, J. M. Hernandez, S. Paik, D. Nguyen, B. Gitlitz, P. I. Shintaku, J. de Kernion, et al.
Induction of G250-targeted and T-Cell-mediated Antitumor Activity against Renal Cell Carcinoma Using a Chimeric Fusion Protein Consisting of G250 and Granulocyte/Monocyte-Colony Stimulating Factor
Cancer Res., November 1, 2001; 61(21): 7925 - 7933.
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