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Clinical Trials |
Division of Immunobiology, National Institute for Biological Standards and Control, Hertfordshire EN6 3QG, United Kingdom [M. W., C. B., R. G-D., R. T.], and Department of Oncology, Karolinska Hospital, Stockholm 17176, Sweden [A-L. H. S., P. R., M. L., H. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20,000 and Mr 30,000. Therefore, in sera from patients receiving product Y, the antibody specificity appeared to be directed not only against GM-CSF but also against non-product-related host cell contaminants. This study shows that GM-CSF products used for therapy are potentially immunogenic and generate antibodies to GM-CSF and/or other non-product-related contaminants. However, only antibodies that neutralize the biological activity of GM-CSF compromise therapeutic efficacy of the cytokine. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the major concerns with the therapeutic administration of cytokines like GM-CSF is the development of antibodies in recipients (10) . Thus far, reports on induction of antibodies in recipients of GM-CSF are scarce because most clinical use of the cytokine has been in patients who are purposely immunosuppressed as part of their therapeutic strategy (11 , 12) . In an attempt to address the issue of immunogenicity of cytokines and whether the development of antibodies diminishes the clinical efficacy of the cytokine product, we have undertaken studies relating to therapeutic use of GM-CSF in nonimmunosuppressed patients. In this study, we have also assessed the clinical consequences of the development of neutralizing and nonneutralizing GM-CSF antibodies in two groups of patients with metastatic CRC receiving two different Escherichia coli-derived GM-CSF products as part of their combination therapy protocol of GM-CSF (with IL-2 in some patients) and a colon carcinoma (CRC)-reactive monoclonal antibody in the absence of any concomitant chemotherapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60 years (range, 36–73 years) entered the three trials (see below). No chemotherapy or immunotherapy was administered for at least 2 months before study entry. Although some patients were untreated, some had received preoperative irradiation or various chemotherapy regimens 2–14 months prior to initiation of the trials.
Treatment Schedule.
In all three trials, E. coli-derived recombinant human GM-CSF was administered s.c. to patients at a dose of 250 mg/m2/day for 10 days. Four cycles were administered, and the treatment cycles were repeated every fourth week. On day 3, 400 mg of colon carcinoma-reactive mouse monoclonal antibody, 17-1A (Trials 1 and 2) or a chimeric antibody (human/mouse) based on 17-1A (Trial 3) was infused i.v. for 60 min. In Trial 2, patients also received rDNA derived human IL-2 produced in E. coli (specific activity, 1.8 x 107 IU/mg protein) at a dose of 2.4 x 106 units/m2 s.c. twice daily for 10 days. Two GM-CSF products prepared by different manufacturers were used in the trials. In Trial 1, patients received GM-CSF product X (Behringwerke, Marburg, Germany; specific activity, 5 x 107 IU/mg protein), whereas in Trials 2 and 3, patients received GM-CSF product Y (Leucomax, Schering-Plough Ltd. Co., Cork, Ireland; specific activity, 1.2–5 x 108 IU/mg protein).
Blood Cell Counts.
The total number of WBCs were estimated by microscopy. Two hundred cells were counted. The percentage of WBC subsets was determined by differential count analysis using May-Grunwald and Giemsa staining. Analysis of the various blood cell counts was carried out using log-transformed values, and the variabilities have been expressed as geometric coefficients of variation (13)
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Cytokine Preparations.
Recombinant DNA-derived human GM-CSF preparations derived from E. coli, yeast, and CHO expression systems were obtained from Schering Plough Corp. (Kenilworth, NJ), Immunex Corp. (Seattle, WA), Sandoz, Ltd. (Vienna, Austria), and Behringwerke AG. These were used for the analysis of the patients’ sera. Recombinant DNA-derived human IL-2 produced in E. coli was provided by Chiron Corp. (Emeryville, CA).
Serum Sampling.
For analysis of anti-GM-CSF, venous blood was collected in sterile tubes; then the serum was separated and stored at -70°C until assayed.
Binding Assay for Detection of GM-CSF Antibodies.
A solid phase indirect ELISA was used to detect GM-CSF binding antibodies in the serum samples from patients. Briefly, flat-bottomed microtiter plates (Maxisorp; Nunc) were incubated at 4°C overnight with 100 µl/well of rhGM-CSF (5.0 µg/ml) in PBS, pH 7.0. The plates were then blocked with 5% milk powder in PBS for at least 30 min at room temperature. Serum samples were diluted 1:20 in 5% milk/PBS, and 100 µl of the samples were then added to microtiter wells in duplicate and incubated overnight at 4°C. The wells were washed extensively with 5% milk/PBS and incubated with 100 µl of horse radish peroxidase conjugated anti-human IgG (1:1000 dilution in 5% milk/PBS; Sigma Chemical Co.) for 2 h at room temperature. After washing five times with PBS containing 0.05% Tween 20, 100 µl of tetramethybenzidine substrate were added to each well; the plates were incubated for 30 min at room temperature, and the enzyme reaction was terminated by the addition of 100 µl of 2 M sulfuric acid. The absorbance of the wells was determined at 450 nm using an ELISA processor. An absorbance value of 0.4 was chosen as the cutoff value in most experiments; this value was found to be higher than the absorbance value noted with serum samples taken from all patients prior to initiation of GM-CSF therapy.
TF-1 Assay for Detection of Neutralizing GM-CSF Antibodies in Sera.
The biological activity of GM-CSF was determined using a bioassay based on the TF-1 cell line, which proliferates in response to GM-CSF (14)
. Briefly, a dilution series of the various rhGM-CSF preparations and the WHO International Standard for rhGM-CSF (88/646) were prepared in 100-µl volumes in 96-well microtiter plates. Exponentially growing TF-1 cells were washed three times, resuspended to a concentration of 105/ml in RPMI 1640 containing 5% FCS and added in 100-µl aliquots to each well. The plates were incubated for 48 h, pulsed for 4 h with 0.5 µCi/well [3H]thymidine, harvested, and the radioactivity incorporated into DNA estimated by scintillation counting (15)
. For neutralization assays, a 2-fold dilution series giving a final 1:20 to 1:2560 dilution of the patients’ sera was preincubated with the various GM-CSF preparations for 1 h at 37°C prior to the addition of cells. Volumes of serum required to neutralize the activity of 1 IU of WHO International Standard for GM-CSF (88/646) were derived using serum ED50 responses obtained from fitting common asymptotes and slope for all sera.
Immunoblotting of GM-CSF Antibodies.
SDS-polyacrylamide electrophoresis under nonreducing conditions was carried out using 12.5% total acrylamide gels (2 µg of protein was loaded per track; Ref. 16
). Samples (GM-CSF protein or pelleted E. coli) were heated at 100°C in sample buffer for 5 min before electrophoresis. The separated proteins were transferred to nitrocellulose membranes, and the membranes were blocked using a solution of 5% (w/v) milk powder in PBS for 30 min on a rotary shaker. The blots were then incubated with serum samples (approximate dilution, 1:200 in PBS/milk) or a polyclonal sheep antibody to human GM-CSF (in-house reagent included as a positive control at a dilution of 1:2000 in PBS/milk in all experiments) overnight at room temperature on a rotary shaker, washed five times with PBS/milk, and further incubated with horseradish peroxidase-conjugated anti-immunoglobulin (of appropriate species specificity, e.g., anti-human; Sigma Chemical Co.) at a dilution of 1:2000 in PBS/milk solution for 1 h on a rotary shaker. The blots were finally washed five times with PBS/0.05% Tween 20, and the immunoreactive protein bands were visualized using the enhanced chemiluminescence reagents obtained from Amersham (Buckinghamshire, United Kingdom).
Adsorption of Sera Using E. coli Spheroplasts.
E. coli K12 strain used for expression of GM-CSF product Y was grown in 500 ml of Luria Broth medium for 5 days at 22°C (17)
. The cell suspension was harvested by centrifugation at 6000 x g for 20 min. The pellet was resuspended in 20 ml of ice-cold 0.5 M sucrose, 100 mM Tris-HCl, and 1 mM EDTA (pH 8.0) and kept on ice for 30 min (17)
. The spheroplast suspension was harvested by centrifugation at 12,000 x g for 10 min, and the pellet was resuspended in 2 ml of PBS. For adsorption of sera, 200 µl of the spheroplast suspension were added to 100 µl of serum and 100 µl of PBS and continuously agitated on a windmill shaker for 3 h at 4°C. The mixture was then clarified in a microfuge for 2 min, the supernatant was removed and incubated with nitrocellulose strips overnight at room temperature, washed with PBS/milk, and finally incubated with horseradish peroxidase-conjugated anti-species antibody and immunoreactive proteins identified using the procedure described in the immunoblotting section.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Approximately 74% of patients treated with product Y (72% in Trial 2 and 75% in Trial 3) had developed GM-CSF-reactive antibodies, using solid phase binding assays at the end of cycle IV, but the incidence was higher in patients treated with product X (Trial 1). In the latter trial (as shown previously), 95% of the tested patients developed GM-CSF antibodies (10
, 18)
. Thus, in total, 28 of the 38 tested patients receiving product Y at the completion of therapy produced GM-CSF-reactive antibodies in comparison with 19 of the 20 patients treated with product X (Table 1)
. The induction of binding antibodies was evident in both groups at the end of either cycle 2 or 3 and was not related to the total dose of GM-CSF administered to the patients (Table 2)
. Detectable GM-CSF antibody disappeared at about 30 weeks after the last injection in Trial 1 patients, but for Trials 2 and 3, there was no evidence of any GM-CSF antibodies at 12 weeks after therapy.
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. The difference in number of patients with regard to induction of neutralizing anti-GM-CSF antibodies between the two groups was statistically highly significant (P < 0. 001).
Cross-Reactivity of GM-CSF Antibodies with Other Recombinant Forms of GM-CSF.
In additional experiments, we evaluated the capacity of antibodies in patient’s sera to bind different GM-CSF preparations produced using different expression systems. Results showed that, in nearly all cases, serum from patients who developed binding antibodies was not only capable of binding the recombinant DNA E. coli-derived GM-CSF preparation used for therapy but also other GM-CSF preparations derived using other expression systems such as CHO cells, yeast, and an rDNA-derived E. coli product produced by a different manufacturer (data not shown).
Induction of Antibodies against IL-2 (Trial 2 Only).
In Trial 2, in which IL-2 was included in the treatment, 6 of the 19 patients studied produced antibodies that recognized IL-2. Only 1 of these patients produced antibodies that neutralized the biological activity of the cytokine, using the CTLL-2 cell line-based bioassay (15)
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Clinical Effects.
In all patients, the total number of leukocytes, neutrophils, eosinophils, lymphocytes, and monocytes were assessed at the beginning (day 1) and end (day 10) of each treatment cycle. Statistical analysis of the data showed significant differences in the total number of leukocytes, neutrophils, and eosinophils at day 10 of cycles 3 and 4 in the blood of Trial 1 patients with neutralizing antibodies in comparison with the cohort of patients that had only GM-CSF binding antibodies and no neutralizing antibodies against GM-CSF (18)
. The latter cohort showed no impaired response to therapy compared with patients with neutralizing antibodies who demonstrated a marked reduction in numbers of leukocytes and neutrophils (with eosinophils and lymphocytes, the decline in numbers was not as pronounced but still clearly apparent) between days 1 and 10 of cycle 4 compared with days 1 and 10 of cycle 1 of the treatment regimen. However, cell numbers were unaffected in the Trial 1 patients who either did not develop antibodies to GM-CSF or developed GM-CSF binding (i.e., nonneutralizing) antibodies only (18)
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In patients treated with product Y (Trials 2 and 3), GM-CSF induced considerable increase in the total number of leukocytes during days 1–10 of all cycles of therapy (Table 3)
. Numbers of monocytes and neutrophils did not differ significantly between patients in the two trials. This was observed irrespective of the development of antibodies. However, levels of lymphocytes were significantly higher in patients in Trial 2 compared with those in Trial 3 (P < 0.05), presumably due to the effect of the IL-2 used in Trial 2. Levels of eosinophils in Trial 3 patients were higher at later stages in the cycles of treatment than for Trial 2 patients (Table 3)
. There was no significant diminution in cell number with progression of therapy, as evident with cell counts seen during cycles 3 and 4 (Table 3)
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Immunochemical Characterization of Antisera.
To assess the binding characteristics of the GM-CSF antibodies produced in patients, we conducted experiments using immunoblotting. Fig. 1
demonstrates typical results obtained using immunoblotting of the two GM-CSF products X and Y with sera from patients from Trials 1, 2, and 3 and a specific sheep anti-GM-CSF serum. We found that sera from Trial 1 patients, irrespective of their neutralizing capacity, demonstrated strong recognition of the GM-CSF protein (Table 2A)
, which migrated as three close bands at Mr 15,000 as shown in Fig. 1B
. These bands were also revealed using a specific anti-GM-CSF serum as in Fig. 1, A and C
. However, sera from Trials 2 and 3 patients showed varied recognition of GM-CSF (strong to very weak) but also bound two additional proteins of Mr 20,000 and Mr 30,000 in product Y only (Fig. 1, D and E
, Lane 2). The recognition of these Mr 20,000 and Mr 30,000 proteins did not occur with the other E. coli-derived GM-CSF preparation, product X (Fig. 1, D and E
, Lane 1).
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shows the typical binding pattern obtained by immunoblotting of three different GM-CSF preparations using serum from a Trial 3 patient. The diffuse band in Fig. 2
, Lanes 1 and 2, is characteristic of the highly glycosylated, CHO cell-derived GM-CSF and the aberrantly glycosylated yeast preparation. The pattern is quite distinct from that obtained with the E. coli preparation shown in Fig. 2
, Lane 3. We found that serum from this patient recognized GM-CSF protein in the three different preparations, but the bands at Mr 20,000 and Mr 30,000 were only evident with E. coli-derived product Y (Fig. 2
, Lane 3), suggesting the presence of a contaminant (unrelated to GM-CSF) in this product. A similar profile was seen with other sera from patients in Trials 2 and 3. In patients treated with product Y, therefore, antibodies appeared to be directed not only against GM-CSF but also against the other two other non-product-related contaminants (Table 2B and 2C)
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shows the results from an immunoblot using serum from a Trial 3 patient posttherapy (A), from the same patient prior to therapy (B), and using a specific anti-GM-CSF serum (C). Prior to initiation of treatment, there was no evidence of any immunoreactivity with product Y (Fig. 3B
, Lane 1), but there was immunoreactivity with high molecular weight proteins in the lysate of the E. coli K12 strain used for expression of product Y (Fig. 3B
, Lane 2). After GM-CSF treatment, however, immunoreactive bands corresponding to E. coli proteins at Mr 20,000 and Mr 30,000 were detected with both product Y (Fig. 3A
, Lane 1) and the E. coli lysate (Fig. 3A
, Lane 2). These results suggest that antibodies to the Mr 20,000 and Mr 30,000 E. coli proteins are induced in patients after therapy with product Y.
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, Lanes 2) completely eliminated the immunoreactivity at Mr 20,000 and Mr 30,000 otherwise detectable with the nonadsorbed serum (Fig. 4, A and B
, Lanes 1), confirming the identity of the Mr 20,000 and Mr 30,000 bands as non-product-related E. coli proteins. On the basis of this data (Fig. 4)
, it appears that GM-CSF product Y contains highly immunogenic E. coli-derived proteins of Mr 20,000 and Mr 30,000.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, are associated with a high incidence of antibody formation (up to 40%). Several studies have reported that patients undergoing therapy with IFN- products develop antibodies that bind the protein and in some instances also neutralize the biological activity of IFN- (20, 21, 22, 23, 24, 25)
. It is conceivable, therefore, that development of such antibodies is one of the reasons for diminished clinical response and perhaps resistance to further therapy with the particular IFN- product or even other IFN preparations, depending on the specificity of the induced antibodies (20
, 24, 25, 26, 27)
. In contrast, some studies have suggested that the development of IFN antibodies has no adverse effects on therapy (28
, 29)
. It has been reported that antibody development is more prevalent in patients treated with rDNA-derived IFN- (23
, 25)
than in those receiving multicomponent IFN products derived from human cells or cell lines (23, 24, 25, 26, 27, 28, 29, 30)
. Therefore, the clinical significance of antibodies, particularly those capable of neutralization, in patients on cytokine therapy needs to be carefully investigated (28)
. Identification of antibodies, in particular neutralizing antibodies, in sera of patients during cytokine-induced therapy is obviously important because it can predict the ability of the induced antibodies to significantly compromise the clinical response to cytokine therapy. To date, information available on immunogenicity of GM-CSF in patients undergoing therapy with the cytokine is scant, often difficult to interpret and obtained from clinical trials using different forms of GM-CSF in immunocompromised patients with underlying disease or advanced malignancies and on intensive chemotherapy, consequently compromising the ability of these individuals to mount an effective immune response. Gribben et al. (11) reported that of 16 patients with malignancies receiving GM-CSF therapy but pretreated with or on intense chemotherapy, 4 developed GM-CSF-reactive antibodies after infusion of yeast-expressed GM-CSF. In a study by Thompson et al. (12) , it was reported that of 16 patients with myelodysplastic syndrome receiving GM-CSF subsequent to immunosuppressive chemotherapy, only 1 developed a low titer of GM-CSF antibodies. In another study, of eight multiple myeloma patients on heavy chemotherapy, only one patient developed GM-CSF antibodies after undergoing treatment with E. coli-derived GM-CSF (10) . This low incidence of antibody formation in immunosuppressed patients receiving GM-CSF therapy for bone marrow reconstitution clearly contrasts with the development of a significant proportion of both GM-CSF-reactive (95%) and GM-CSF-neutralizing antibodies (45%) in 20 nonimmunocompromised patients receiving therapy with E. coli-derived GM-CSF (18) . However, the incidence of induction and characteristics of antibodies against GM-CSF appears to be product related because this study shows that an rDNA E. coli-derived product produced by a different manufacturer was less immunogenic. With this product (product Y) used at substantially higher doses in comparable trials to those carried out previously with product X (specific activity is different for the two products), 74% (28 of 38) of patients developed antibodies that bound to the product using the binding assay cutoff value approach (based on elimination of all pretrial serum samples as being considered positive) for discrimination of positives from negatives adopted for patients treated with product X. None of these was able to neutralize the biological activity of the cytokine. Unexpectedly, the specificity of the antibodies produced by patients receiving the different products differed dramatically. All antibodies produced by recipients of product X bound to GM-CSF or product-related molecular species; however, all antibodies produced by patients receiving product Y bound to two non-product-related E. coli-derived proteins, irrespective of whether these patients also produced antibodies against authentic GM-CSF proteins. Only 15 (38%) of patients treated with product Y produced detectable antibodies against GM-CSF. Using the sensitive immunoblotting method, antibodies against E. coli proteins could be detected in serum from all patients except one, who received product Y although the intensity of staining varied considerably from very strong to very weak. The discrepancy between antibody detected by immunoblotting and the binding immunoassay data reflects the low level of E. coli proteins in the final product and the adoption of an "arbitrary" cutoff value for determining results based on elimination of all pretrial serum samples as being considered positive.
The reason for the potent immunogenicity of the two E coli-derived proteins in recipients of product Y was unclear. Immunoblotting using pretreatment sera or serum from normal individuals failed to detect antibodies to either protein, although several higher molecular weight E. coli proteins were recognized. The two proteins were trace contaminants present in product Y (but not product X) requiring sensitive silver staining of polyacrylamide gel electrophoretograms for their detection. Their presence on E. coli spheroplasts strongly suggests that they are located on the surface of the bacterial inner membrane and penetrate the periplasmic space of the bacterium (into which many rDNA-produced molecules are secreted), and this would also explain the absence of antibodies capable of recognizing these proteins in individuals who have not received product Y. Their immunogenicity may be due to intrinsic properties and/or adjuvant effects due to GM-CSF (31 , 32) . The latter effect might also explain the apparently greater immunogenicity of GM-CSF compared with other cytokines, although the appreciable level of production of antibodies that recognized IL-2 in Trial 2 (33%) could imply that cytokine antibody development in nonimmunocompromised patients receiving multiple doses of cytokine is more frequent than considered previously. In any case, the greater immunogenicity of GM-CSF proteins in product X compared with product Y shows that GM-CSF products per se differ in some aspects important for antibody development. It is possible that differences in the amino acid sequence (product X differs from the human gene product and from product Y by the addition of a proline residue at the NH2 terminus), production and purification procedures for the different products (33 , 34) could influence their immunogenicity.
The clinical importance of induction of antibodies against GM-CSF products depends on the antibody specificities and characteristics. As we have shown previously, only antibodies that neutralize the biological activity of GM-CSF appear to compromise clinical response (Trial 1; Ref. 18 ). Additional new data obtained from Trials 1, 2, and 3 included in this report confirm this in vivo. Nonneutralizing antibodies that bind GM-CSF do not seem to reduce clinical response, even if they are present at a relatively high titer. The significance of nonneutralizing antibodies against the cytokine is therefore unclear. It is even more difficult to assess the consequences of development of antibodies against the two E. coli proteins. These will obviously not inhibit GM-CSF activity and so do not compromise the clinical efficacy of therapy with the cytokine (our data clearly confirm this). It seems plausible that anaphylactoid-type adverse effects might be mediated by such antibodies if they react with sufficient antigen, but this seems unlikely considering the non-surface location of the antigens on E. coli and the small amount of the proteins present in product Y; no such reactions were noted in the study. More importantly, such antibodies could be confused with antibodies that recognize "genuine" GM-CSF protein in therapeutic products, and this is taken as evidence that therapy may be compromised. This mistake could easily occur if binding assays such as ELISAs are solely used for antibody detection (as is often the case).
In conclusion, this study clearly demonstrates that GM-CSF preparations show important product-related differences in immunogenicity. When administered to nonimmunosuppressed patients, the cytokine seems significantly immunogenic, and antibodies can be produced that bind GM-CSF and neutralize the biological activity of the cytokine, bind but do not neutralize activity, and/or bind to non-GM-CSF contaminants. Only antibodies that neutralize the biological activity of GM-CSF compromise clinical response. A clear correlation of neutralization assessed by an in vitro bioassay with impaired clinical response was observed. No obvious correlation between neutralizing capacity and binding titer (using ELISA) was noted. The use of bioassays to assess antibody induction in patients, therefore, provides a useful assessment of potential antibody-mediated inhibition of therapeutic response. However, detection of antibodies using binding assays such as ELISAs does not provide comparable data, and results obtained using such methods alone may be misleading as an indicator of a potentially impaired clinical response to GM-CSF therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by grants from the Swedish Cancer Society and the Cancer Society in Stockholm. The study was approved by the Ethics Committee of the Karolinska Institute. 
2 To whom requests for reprints should be addressed, at Division of Immunobiology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom. Phone: 44 01707 654753; Fax: 44 01707 650223; Email: mwadhwa{at}nibsc.ac.uk
3 The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; rhGM-CSF, recombinant human GM-CSF; IL, interleukin; CRC, colorectal carcinoma; CHO, Chinese hamster ovary.
Received 12/ 2/98; revised 2/ 5/99; accepted 2/11/99.
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