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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Cancer Res. Author manuscript; available in PMC Sep 15, 2010.
Published in final edited form as:
PMCID: PMC2745522
NIHMSID: NIHMS131782
Immunogenicity of the Hu14.18-IL2 Immunocytokine Molecule in Adults with Melanoma and Children with Neuroblastoma
Jacquelyn A. Hank,* Jacek Gan,* Hyunji Ryu,* Amy Ostendorf,* Michael C. Stauder,* Adam Sternberg,* Mark Albertini,* Kin-Ming Lo,** Stephen D. Gillies,**# Jens Eickhoff,* and Paul M. Sondel*
*The University of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison WI
**The EMD-Lexigen Research Center, Billerica MA+
+EMD Pharmaceuticals Inc., Durham, NC (now EMD Serono, Inc.).
#Currently employed by Provenance Biopharmaceuticals Corp.
Purpose
Immunocytokine (IC) hu14.18-IL2 is a fusion protein of humanized anti-disialoganglioside (GD2) antibody (hu14.18) and interleukin-2. Sixty-one melanoma and neuroblastoma patients received IC in Phase I/Ib studies. Patient sera were examined in enzyme-linked immunosorbent assay (ELISA) to determine if an anti-IC antibody response occurred during treatment.
Experimental Design
Serum was assayed for anti-idiotypic antibody based on ability to bridge biotinylated hu14.18 to plate-bound hu14.18 and ability to inhibit binding of hu14.18 to GD2 antigen and/or murine anti-id antibody. ELISA was also used to detect antibodies to the Fc-IL2 end of hu14.18-IL2.
Results
Thirty-two patients (pts) (52%) developed an anti-id antibody response (OD > 0.7) in the bridge ELISA. Twelve pts (20%) had an intermediate response while 17pts (28%) were negative (OD < 0.3). The development of antibody to hu14.18-IL2 detected in the bridge ELISA was not related to the dose of Hu14.18-IL2. Twenty of 33 adult pts (61%) demonstrated anti-id based on binding inhibition ELISA. The anti-id response was inversely correlated (p<0.002) with IC measured during the second course of treatment, indicating that development of anti-id antibodies interfered with detection of circulating Hu14.18-IL2. All pts developed some inhibitory activity in the binding inhibition assay designed to detect antibodies to the Fc-IL2 region of the IC. There was a positive correlation between the peak serum level of IC in course 1 and the anti-Fc-IL2 response.
Conclusions
Pts treated with hu14.18-IL2 developed anti-idiotypic antibodies and anti Fc-IL2 antibodies. No association was seen between development of anti-IC antibodies and clinical toxicity.
In an effort to improve anti-tumor effects with IL-2 (1) or mAb (2) alone, or combined treatment with the individual components (3-7), an immunocytokine (IC) (8,9) was created which contains the tumor reactive 14.18 mAb linked to IL-2 at the carboxy terminus of each IgG1 heavy chain. The proposed mechanism of action is localization to tumor via recognition of tumor associated GD2 disialoganglioside (10-13). Localization of IC facilitates activation of natural killer (NK) cells through Fc and IL-2 receptors (14) and activation of T cells through IL-2 receptors (15). NK cells mediate cytolytic activity via antibody dependent cellular cytotoxicity, (ADCC) and non-MHC restricted cytotoxicity (9). In some preclinical models, tumor antigen specific T cell memory is also induced (15,16). Clinical reports for separate Phase I studies treating melanoma and neuroblastoma patients with this IC were recently published (14,17). The present study was designed to determine if pts receiving the IC developed an immune response to the IC. We monitored pts for development of antibody to the IC. Adult MEL pts with responding or stable disease were eligible to receive a second course of IC (14). Pediatric NBL pts with stable or responding disease were eligible to receive up to 4 or 6 courses of IC respectively (17). We established ELISAs to detect antibodies specific for the two separate functional ends of the IC. Antibodies against the idiotypic (id) determinant (18) and against the carboxy terminus of the IgG heavy chain where IL2 is linked (Fc-IL2 end) were detected. These antibodies could potentially interfere with the proposed functions of the IC. An anti-idiotypic (anti-id) antibody might prevent the IC from targeting to tumor (18). An antibody against the Fc-IL2 end of the IC (anti-Fc-IL2) might interfere with immune activation facilitated through IL-2. We report here on the occurrence, frequency, and potential immunological effects of the antibody response to hu14.18-IL2.
Hu14.18-IL2
IC (EMD 273063) was provided by EMD Pharmaceuticals Inc., Durham, NC (now EMD Serono, Inc.). One mg of IC contains 3 × 106 IU of IL2 (19) and 0.8 mg of the hu14.18 antibody.
Study Design
These phase I trials were nonrandomized dose escalation studies. Initial clinical and immunological results were previously reported (14, 17). Briefly, hu14.18-IL2 was given as a 4 hour IV infusion on days 1, 2 and 3 of each 28 day treatment course. Adult pts received up to two courses and pediatric pts received up to 6 courses of IC. Unless otherwise indicated, serum samples were taken with morning blood draws, prior to administration of IC. The day and course for blood samples are identified as follows: C1D1 = course 1, day 1; C3D8 = course 3, day 8. Peak IC serum levels were determined from blood obtained within ½ hour of completing the IC infusion.
Cell lines
M21 (GD-2 positive melanoma) (14,17) and IL-2 receptor positive RL-12 (subline of NKL-human leukemia obtained from Dr. Paul Leibson of the Mayo Clinic, Rochester MN) (20) were maintained as previously described.
Enzyme-linked immunosorbent assays (ELISAs)
SIL-2Rα was measured by (Immunotech, Marseilles, France) ELISA kit.
Detection of IC
Measurement of IC in patients' sera by ELISA was performed as previously described (18,21,22).
Detection of anti-IC antibodies
The humanized 14.18-IL2 has two types of immunogenic epitopes recognized as foreign by some patients. The anti-GD2 idiotypic determinant and the determinants created where IL-2 is directly linked to the carboxy terminus of IgG heavy chains, referred to as Fc-IL2. We have created ELISAs which allow the detection of antibodies to these determinants (Fig1).
Figure 1
Figure 1
ELISA methods to detect antibodies to the hu14.18-IL2 IC. A) Anti Id-Bridge detects anti-idiotypic antibody in pt serum bridging biotinylated hu14.18 antibody to plate bound 14.18 antibody. B) Anti-IC – Binding inhibition (GD2) detects anti-id (more ...)
Fig. 1A
Anti Id-Bridge to detect anti-14.18 Idiotypic antibody is similar to our previously published “bridging” method detecting anti-14.18 chimeric antibody (23). Serum samples obtained prior to therapy from all patients were negative in this assay (OD < 0.3).
Fig. 1B
Anti IC-Binding inhibition (GD2) for detection of Anti-IC antibodies using GD2 coated plates (21). Hu14.18-IL2 binds to GD2 and is detected using biotinylated anti-IL2 antibodies. When patient serum containing anti-id antibody is combined with IC prior to adding to GD2 coated plates, the anti-id antibody inhibits the 14.18 Ab component of the IC from binding to GD2. In addition, when patient serum contains antibodies specific for the Fc-IL2 determinant, this antibody binds to the IC and inhibits the biotinylated anti-IL2 antibody from detecting the IC. Thus this ELISA detects anti-IC antibodies against both idiotypic and Fc-IL2 determinants. Briefly, purified disialoganglioside GD2 (Advanced ImmunoChemical Inc., Long Beach CA) suspended in 100% ethanol was absorbed to 96-well flat-bottom polystyrene plates (Falcon #3915, Fisher Scientific, Hanover Park, IL) at a concentration of 250 pM. Hu14.18-IL2 was diluted to 250 ng/ml and further diluted 1:2 with patient serum. This mixture was incubated at room temperature for one-half hour and 100μl/well added to duplicate wells and incubated for 2 hours at 37°C. Hu14.18-IL2 bound to GD2 was detected using biotinylated goat-anti-human-IL2-antibody (8.33 ng/ml) (R&D Systems, Minneapolis, MN), followed by ExtrAvidin-HRP conjugate (2μg/ml) (SIGMA-Aldrich Chemicals. St. Louis, MO) and addition of TMB substrate (DAKO Chemicals, Carpinteria CA). The reaction was stopped using 50μl/well of 2N H2SO4. The absorbance (OD 450/570) was used to determined the amount of hu14.18-IL2 detected interpolating to a standard curve. The % inhibition was then calculated as:
equation M1
The specificity of the anti-IC antibodies detected in the binding inhibition assays was assured by comparing IC detected using pretreatment serum to IC detected using serum obtained at times following treatment.
Fig. 1C
Anti IC-Binding inhibition (1A7) for detection of Anti-IC antibodies using 1A7 coated plates. This assay is identical to the anti-IC binding inhibition assay described in the paragraph above (for Fig.1B) with the exception that the capture molecule is the murine 1A7 antibody. This antibody, specific for the idiotypic determinant of the 14.18 anti-GD2 antibody binds to the 14.18 mAb, and thus serves as a mimic for GD2 (24).
Fig. 1D
Anti Id Binding inhibition for detection of Anti-Id antibody by using 1A7 mAb coated plates and biotinylated hu14.18 mAb. The 1A7 mAb resembles the GD2 antigen and will capture or bind biotinylated hu14.18 Ab. The bound biotinylated 14.18 can then be quantified using the same biotin-avidin enzyme system used in the bridging assay described above (Fig.1A). Briefly, C8 Maxisorp microtiter plates (Nunc; Denmark) are coated overnight at 4°C with 120 μl of 2 μg/ml 1A7. Serum samples are diluted 1:5 with a solution of 3.1 ng/ml biotinylated hu14.18 and added to plates (100 μl/well). The plates are incubated overnight at 4° C and bound 14.18 determined using the same biotin-ExtrAvidin-HRP enzyme system used above. Results are presented as “% inhibition”, where the amount of hu14.18 detected in pre-treatment serum is defined as 0% inhibition for that patient: and % inhibition is calculated as for Fig. 1B.
Fig 1E
Anti Fc-IL2 Binding inhibition for detection of antibody to the Fc-IL2 region of the IC using anti-IL2 coated plates and biotinylated Fc-IL2. Briefly, microtiter plates (Nunc; Denmark) are coated overnight at 4°C with 120 μl of 2 μg/ml neutralizing rat monoclonal anti-human IL-2 antibody MQ1-17H12 (BD Pharmingen). Serum samples are diluted 1:5 with a solution of 5 ng/ml biotinylated hu14.18 Fc-IL2 fragment and added to plates at 100 μl/well. The plates are incubated overnight at 4° C and bound Fc-IL2 determined using enzyme system described above. Any decrease in the detected amount of Fc-IL2 from that expected reflects the presence of inhibitory activity (anti-Fc-IL2 antibody). Results are presented as “% inhibition”, where the amount of Fc-IL2 detected in pre-treatment serum is defined as 0% inhibition for that patient, and % inhibition is calculated as for assays B and D above.
Statistical Analysis
The association between anti-id activity and IC dose used Pearson's correlation analysis. The comparison of peak serum levels of hu14.18-IL2 between courses used Wilcoxon Signed Rank test. An exact McNemar's test was used to compare anti-id and anti-Fc-IL2 response rates between courses. Logistic regression analysis was performed to assess the dose response relationship between IC dose and anti-FC-IL2 response. Chi-square analysis was used to evaluate the association between anti-IC antibodies and clinical toxicities. A two-sided significance level of 0.05 was used for all statistical tests.
Detecting and quantifying anti-id responses
The anti id-bridge assay, based on our previously developed human anti-chimeric antibody (HACA) assay (23) was established to specifically see how well patients' serum specimens (obtained at ≥ 3 times per 4 week treatment course) could bridge biotinylated hu14.18 mAb to hu14.18 bound to the plate (Figure 1A).
Of 61 adult and pediatric pts evaluated in these Phase I dose escalation studies, reflecting 126 courses of hu14.18-IL2 treatment, 28% were negative, 20% showed a minimal response and 52% had a positive anti-id response with at least one specimen showing an OD reading > 0.7 No pretreatment samples were positive and there was no correlation of anti-id activity and IC dose. The “anti-id” specificity of this assay was demonstrated by showing that serum samples from patients that were able to “bridge” biotinylated hu14.18 mAb to hu14.18 bound to the plate could also bridge biotinylated hu14.18 mAb to a plate pre-labeled with ch14.18 mAb, but not to plates pre-labeled with Rituxan, Herceptin, KS1/4, R24 or UPC-10 humanized or murine mAbs (data not shown).
We next determined when the peak anti-id responses were detected. The adult study included 19 pts who received two courses of IC. Seven pts did not develop any “bridging antibody,” while 12 demonstrated a detectable anti-id response. Five of these 12 demonstrated their highest anti-id activity in course one, three in course two, and four had similar levels of anti-id bridging antibodies during course one and two. In the pediatric study there were 20 pts who received more than one treatment course. Three of these pts did not develop a bridging antibody (one pt with 3 courses and 2 pts with 4 courses). Seventeen developed detectable anti-id, 9 had their highest OD value in their first course, four during their final course and 4 during a course between their first and final courses. Thus it appears that for this bridging anti-id assay, the highest values are more often observed after the first course than the last. Of 29 pts in the 2 studies that received more than 1 course and developed an anti-id response, 14 had their highest OD value during course 1, while only 7 had their highest value after the last course. This indicates that, for most pts, continued exposure to IC does not continue to “boost” the anti-id response detected in this bridge assay.
The influence of anti-IC antibody on IC binding to GD-2
We tested whether the serum samples could inhibit hu14.18-IL2 IC from binding to GD2. We devised a GD2-based anti-IC ELISA (Fig. 1B) (21) where serum from a pt is incubated with a fixed amount of IC to determine if anti-id Ab inhibits binding of hu14.18-IL2 to the plate-bound GD-2. This assay was used for the first 18 MEL pts accrued to the Phase I study. Six pts showed measurable activity in the anti-IC-GD2 binding inhibition ELISA but no activity in the bridge assay (Fig.1A and data not shown). This suggests that this binding inhibition assay (Fig.1B) is more sensitive than the bridge assay, or that it may detect Ab not detectable in the bridge assay. It is possible that some high affinity and/or low level of anti-id antibody that can be detected in this GD2 binding inhibition assay may not be detected in the bridging ELISA, indicating a potential distinction in sensitivity for anti-id detection. The IC structure is such that 2 distinct types of anti-IC antibody might be detected in this GD2 based IC-binding inhibition assay. First, anti-id antibodies could prevent the IC from binding to the plate bound GD2. Second, an Ab against the Fc-IL2 linkage region might interfere with detection of IC when using an anti-IL2 detection Ab (shown schematically in Fig. 1B). To address this, we developed two additional ELISA assays that independently detected anti–id Ab or Ab against the Fc-IL2 linkage component. These are the anti-id binding inhibition (Fig.1D) and anti-Fc-IL2 binding inhibition (Fig.1E) assays.
Anti-idiotypic antibodies
The anti-id binding inhibition assay was designed to detect Ab-specific for the idiotypic determinant. Pts' serum was added to biotinylated hu14.18 mAb, prior to the ELISA capture step (Figure 1D). The capture antibody is the murine 1A7 mAb (24), specific for the 14.18 Ab idiotypic determinant. When pts have an anti-id Ab, it binds to the idiotypic site of the hu14.18 mAb and may compete for the binding of the hu14.18 by the 1A7 plate-bound “capture” mAb, thereby inhibiting the binding of the hu14.18-biotin to the plate (as detected by the ExtrAvidin-HRP system). This assay has been performed on serum from all 61 pts in these Phase I trials. The results from course 1 for all adult MEL pts are shown in Figure 2A. Results are plotted as “% inhibition,” based on how serum obtained after IC treatment compares to pretreatment serum. Each line reflects data from a separate pt. When data from both course 1 and 2 for these 33 adults are combined, 26 patients (79%) showed a statistically significant anti-id response. Serum specimens showing >28% inhibition were considered to show positive responses (above background), based on being greater than the mean inhibition plus 2 SD for the pretreatment serum samples from these same pts in this assay. When results in the anti-id bridge and anti-id binding inhibition assays are compared, for all 126 treatment courses for all 61 pts, 4 distinct reactivity patterns are observed. Nine pts (15%) were negative in both assays. Thirty two pts (52%) developed anti-id activity detected in both bridging and binding inhibition assays. Eight of the 61 pts (13%) developed reactivity in the binding inhibition assay but not in the bridge assay. Twelve pts (20%) were positive in the bridge assay and negative in the binding inhibition assay.
Figure 2
Figure 2
Anti-ID (Assay Fig 1 D) and Anti-Fc-IL2 (Assay Fig 1E) results for C1D0, D8 and D15 for all 33 Adult pts in course 1. Results shown are % inhibition, based on the change in concentration of biotinylated hu14.18 (2A) or biotinylated Fc-IL2 (2B) detected (more ...)
While the magnitude of the anti-id response detected in this binding inhibition assay was not influenced by the dose of hu14.18 administered, it was influenced by the number of courses the individual patients received. This was noted in the pediatric study where 28 pts received from 1-4 courses of hu14.18-IL2. The percentage of pts developing > 50% inhibition in the anti-id binding inhibition assay increased with the number of courses of IC administered (Table 1). This change in reactivity was significant between course 1 and subsequent courses, (course 1 vs course 2, 3 or 4, p= 0.002, p = 0.026, p= 0.007 respectively) as well as when comparing course 1 to courses 2, 3 and 4 combined (p≤ 0.001). This contrasts with the results of the bridging anti-id ELISA, where more pts developed their highest bridging activity during their first course.
Table 1
Table 1
Time course for development of anti Id and anti Fc-IL2 antibodies in pediatric patients
We next asked whether the induction of anti-id antibody as detected by this anti-id binding inhibition ELISA (Fig 1D) would also influence the serum levels of hu14.18-IL2 measured during a second course of treatment. When data from eleven evaluable adult melanoma pts, who received 2 courses of IC at the same dose for each course, were analyzed, those pts that showed greater inhibitory activity in this binding inhibition assay also showed a corresponding decrease in their peak IC serum level measured immediately after finishing the 4 hr infusion on C2D1 compared to their peak level obtained immediately after the 4 hr infusion of the same IC dose on C1D1 (R = -0.83, p = 0.002, data not shown). This indicates that the pts with the strongest anti-id Ab responses show the lowest detectable serum IC levels during the first day of their second course of treatment.
Anti-Fc-IL2 antibodies
A separate “anti-Fc-IL2 binding inhibition” assay was designed to detect antibody specific for the Fc-IL2 linkage component of the IC (Fig.1E). Serum specimens were added to biotinylated Fc-IL2 fragments prior to their capture on the ELISA plate coated with an anti-IL2 Ab, followed by detection with ExtrAvidin-HRP. If the pt made an Ab that bound to this fragment it might interfere with the binding of the Fc-IL2 to the capture anti-IL2 antibody. Data from all 33 adult MEL pts are shown for course 1 in Fig. 2B. C1D8 serum from 32 of 33 patients showed development of detectable inhibitory activity. Table 2 shows representative anti-IC values in the 3 binding inhibition assays (Fig. 1B,C,D) for 2 pts with strong anti-id responses. Separate assays (not shown) evaluate the functional effects, and the specificity, of the anti IC antibodies detected in these ELISA assays, and will be presented in a subsequent report (Hank et. al., in preparation). There was no relationship between the magnitude of the anti-Fc-IL2 response, and the level of hu14.18-IL2 detected in the serum during course 2 (R = 0.272, p = 0.41). A dose-response relationship was identified for this anti-Fc-IL2 response; pts receiving higher doses of IC show stronger anti-Fc-IL2 responses measured on day 8 and day 15 in course 1 (p=0.005, data not shown). Similarly there was a significant positive correlation between the area under the curve for serum concentration of hu14.18-IL2 on C1D1 and the development of anti-Fc-IL2 antibodies. The biological mechanism for these results requires further evaluation, but might suggest that greater doses of hu14.18-IL2 or greater exposures to hu14.18-IL2 were more potent at inducing the anti-Fc-IL2 response.
Table 2
Table 2
Anti-hu14.18-IL2 activity (% inhibition) detected in binding inhibition assays.
Clinical correlations with anti-IC antibody
Data from 2 representative NBL pts receiving 4 courses of hu14.18-IL2, one with and one without an antibody response to the IC, are presented in Fig. 3. Pt 10 generated very little anti-id antibody (Fig. 3A) and the amount of IC detected in the serum was similar for all four courses (serum IC levels were measured on days 1 and 3 of each treatment course; In contrast, Pt 19 developed anti-IC antibodies during the first course of treatment which were evident in the binding inhibition assay by day 8 of course one (Fig. 3B). Anti-id antibodies were also noted in the bridging assay following courses 2, 3 and 4. The serum IC levels detected during courses 2, 3 and 4 were markedly lower than the levels seen in course one, indicating that the anti-IC response was associated with lower detectable IC. The lymphocyte count and serum level of sIL2Rα are both indicators of immune activation induced by IL2 (25). Even though the level of IC detected in serum from patient 19 was lower in courses 3 and 4 than in course 1 (presumably due to the anti-IC response), the lymphocytosis and increase in sIL2Rα in courses 3 and 4 were comparable to that seen in course 1 (Fig. 3D). Thus administration of IC in courses 3 and 4 to patient 19 still resulted in comparable IL2-induced in vivo activation (lymphocytosis and increase in sIL2Rα despite the relative inability to detect circulating IC in the ELISA. Patient 10 generated very little anti-id antibody and values for lymphocyte counts and sIL2Rα increased and remained elevated throughout the 4 courses of therapy (Fig 3C).
Figure 3
Figure 3
Serum IC level measured at multiple time points on days 1 and 3 of each 3 day course of IC for 2 patients who received 4 courses of hu14.18-IL2. The IC level is indicated by the solid lines with each of 10 determinations per infusion indicated by an open (more ...)
As noted above, greater anti-id responses were associated with an inhibition in the detected levels of IC on C2D1, (p< 0.002). Either the circulating anti-id in these pts is removing the IC from the serum, or it is preventing it from being detected in the ELISA. Data from Pt 19 in the pediatric trial (Fig. 3) shows that the anti-id response may be preventing IC detection, as well as removing some IC from the circulation. The serum levels of hu14.18-IL2 immediately after the 4h IC infusion (Fig.3) have been measured on days 1 and 3 for each of the 4 courses.
The levels of anti-id and anti-FcIL2 Ab were evaluated in the MEL study for any relationship with the previously published hu14.18-IL2 induced: a) change in lymphocyte counts; b) change in lymphocyte phenotype [increase in % CD 16+, and CD56+ and decrease in % CD3+ PBMC]; and c) increase in serum sIL2R level (14). No significant correlations were seen between the anti-id response and these 3 parameters. The anti-Fc-IL2 response did not correlate with lymphocyte numbers or phenotype, but pts that induced the strongest anti-Fc-IL2 Ab were also those that showed a greater increase in their sIL2Rα levels in course 2 than in course 1 (p< 0.01). The mechanism for this relationship remains uncertain.
Treatment of adult and pediatric patients with IC induces Ab responses to IC in some but not all patients. Some patients initially make a low level of Ab that does not increase progressively with increasing number of courses. Others make antibody responses that are more detectible with subsequent courses of treatment. This phenomenon was previously reported by Welt et al. who treated 11 pts with advanced colorectal cancer with the humanized IgG1 A33 Ab (26). Of these 11 patients, 3 did not develop an antibody to A33 Ab. The 8 pts developing a “human-anti humanized-antibody” (HAHA) response, were divided into two groups. Five pts developed a Type I HAHA response – which generally developed early, within 2 weeks of treatment and usually resolved within 7 weeks of treatment and did not progress with the number of courses. Three pts developed a Type II HAHA response, characterized by progressively increasing titers of anti-huA33 antibodies. We noted similar trends in anti-IC responses. Some patients developed antibodies which increased in magnitude with the number of courses (i.e.: Pt 19, Fig. 3). Some patients developed detectable levels of anti-IC Ab, but the strength of the response did not continue to increase, or did not appear to affect the level of IC detected in the patient serum following subsequent IC infusions (ie: Pt 10 Fig.3). While it appears that Pt 19 developed a response that is similar to the Type II response described by Welt et al., the significance of this response in these IC treated patients is not yet known, as the generation of an Ab was not associated with increased toxicity upon subsequent infusions of IC.
In Fig 3 (and in data not shown) anti-IC antibodies that are detected in the binding inhibition ELISA limit the detection of IC in patient serum. Of interest are the anti-id antibodies measured by the bridging assay on days immediately following each 3 day course of IC. In Fig 3B, pt 19 shows high OD values corresponding to the levels of anti-id “bridging” antibody at C2D8 and all subsequent time points, except for the striking drop in activity seen on day 4 of courses 3 and 4. These serum samples are obtained 20 hours following the completion of the 3rd IC infusion. As the half-life of the IC is 3.1-3.7h (14, 17), circulating IC should not be influencing the results of the Course 3 and 4 day 4 bridging assay. The major drop in the anti-id antibody seen in courses 3 and 4 on day 4 compared to the day 1 value suggests that the 10 mg/M2 of hu14.18-IL2 infused daily on days 1, 2 and 3 of that course may have “complexed” with the majority of the circulating anti-id antibody, thereby limiting the amount of anti-id antibody that could be detected on day 4. The peak IC serum levels during courses 1 and 2 are approximately 4μg/ml while during courses 3 and 4, serum IC was virtually undetectable. This absence of detectable IC following an IV hu14.18-IL2 infusion suggests that the anti-IC response was able to “neutralize” virtually all of the hu14.18-IL2 given to this patient at that time. Similarly, the anti-id response measured with the bridge ELISA on day 4 of courses 3 and 4 is only ~20% of that seen on day 1 of these same 2 courses, suggesting that infusing hu14.18-IL2 that generated a peak serum level of 4μg/ml during course 1 and 2, was able to “neutralize” most of the circulating anti-IC antibody. This suggests that the serum level of functional anti-IC antibody might also be near 4μg/ml. Because our other methods of anti-id detection require binding inhibition assays, where the inhibiting anti-IC antibody is in excess, it is impossible to determine the actual concentration (i.e. mcg/ml) of anti-IC Ab. The antibody to the Fc-IL2 determinant does not completely inhibit Fc-IL2 binding to anti-IL2 mAb in the ELISA. Unlike the anti-id assays, where % inhibition values were occasionally approaching 100%, % inhibition values in the anti-Fc-IL2 assay rarely exceeded 90% (see table 2). Furthermore anti-Fc-IL2 inhibitory activity detected in these ELISA is readily lost with a 10 fold dilution of the serum sample (data not shown). In contrast, anti-id can completely inhibit specific binding of the hu14.18 antibody to GD2 coated (Fig. 1B) and 1A7 coated (Fig. 1C) ELISA plates. In some pts, serum samples continue to inhibit at dilutions as great as 1:100. However in two pediatric pts with considerable inhibition in the anti-id binding inhibition assay throughout 4 courses of treatment, the serum sample with the most potent inhibition (and the ability to retain inhibitory activity with 1:100 dilution) was noted in the 2nd, rather than 3rd or 4th course (data not shown), suggesting some waning of the anti-id response.
The clinical significance of the anti-id antibodies is yet to be determined. Of these 61 melanoma and neuroblastoma patients, 52 developed antibodies detected in either the anti-id bridge or anti-id binding inhibition assays. Of these 52, 32 patients were positive in both assays, 8 were positive only in the binding inhibition assay and 12 were positive only in the bridge assay. These anti-id antibodies were not associated with an increase in toxicity or allergic phenomena (14, 17). Nor were these anti-id antibodies associated with changes in clinical laboratory markers or markers associated with IL-2 induced immune activation (sIL-2Rα and increase in the lymphocyte count). It is not clear if the amount of anti-IC Ab present in the serum would be sufficient to limit the ability of the IC to target GD2 positive tumors in vivo, and if so, what levels of reactivity in vitro would correspond to meaningful in vivo inhibition of function. Our preliminary data indicate that levels of anti-IC antibody detected for most patients in the ELISA reported here do not necessarily correspond to levels that inhibit IC binding to GD2 or IL2R in conditions designed to simulate the in vivo setting (Hank et al, in preparation). The development of anti-Fc-IL2 Ab also was not associated with an increase in toxicity or change in the lymphocyte count, but there was a significant hu14.18-IL2 dose effect and correlation of anti-Fc-IL2 response with the AUC for hu14.18-IL2. This suggests that greater exposure to hu14.18-IL2 induces more anti-Fc-IL2 antibodies. In addition pts that induced the strongest anti-Fc-IL2 Ab were those that showed a greater increase in their sIL2R levels in course 2 than in course 1. This indicates an association between stronger activation of sIL2R in a pt's 2nd course with induction of a strong anti-Fc-IL2 Ab. The mechanism of this requires further study, as do its clinical implications.
While anti-id antibodies can interfere with the desired antigen binding function of the mAb, at least in vitro, it remains controversial whether an anti-id response might be beneficial or harmful for the therapeutic effect desired in treated pts. In some clinical studies of mAb used as cancer therapy, the presence of an anti-id antibody response, particularly if transient, has been associated with an improved antitumor effect (27-29). This improved effect has been postulated to result from the induction of an “anti-anti-idiotypic” response, designated an “antibody-3 response”, which may have direct antitumor activity. In fact, clinical efforts to induce a potent antitumor response have utilized immunization with anti-idiotype antibodies as vaccines, designed to activate a tumor reactive “antibody-3” response (22, 29). Our initial efforts to detect antibody 3 responses in selected patients showing strong anti-id responses have not revealed antibody 3 activity (data not shown). It remains uncertain whether the induction of the anti-id and anti-Fc-IL2 antibodies might help or interfere with the desired in vivo clinical effect. As there were no clinical responses noted in these Phase I studies, it is impossible to predict the impact of the generation of antibody response to the IC upon clinical outcome. We have just completed phase II trials of this agent for 14 adults with MEL and 39 children with NBL; anti-tumor activity was seen in each study (30, 31). We are now performing the in vitro analyses of anti-IC responses for these 53 patients.
In summary, anti-IC antibodies were formed in most patients. These included HAHA responses to the idiotypic determinant and antibodies specific for a neoantigen created at the Fc-IL2 junction site of the IC molecule. These antibodies do not appear to induce allergic reactions or increase the toxicity of the IC. The in vivo functional significance of this antibody response is yet to be determined. The lymphocytosis and sIL2R levels seen in the face of highly reactive anti-Fc-IL2 antibody, suggest that these anti-IC antibodies do not inhibit IL-2 induced immune activation in vivo. Additional studies are underway to better characterize the in vivo effects of these anti-id and anti-Fc-IL2 antibodies.
Acknowledgments
This work is supported by: NIH CA32685, CA14520, CA87025, CA81403, RR03186, and a grant from the Midwest Athletes for Childhood Cancer Fund. The pediatric Phase I Study (COGADVL0018) was supported by the Children's Oncology Group (COG), and performed by COG phase I institutions.
Footnotes
Statement of Translational Relevance. This paper presents the results of immunologic monitoring of melanoma and neuroblastoma patients receiving hu14.18-IL2 as part of Phase I clinical trials. The discovery that some of these patients develop antibodies to the hu14.18-IL2 molecule will help us in the design of future clinical trials with this molecule.
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