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, ). 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 ). 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 (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 , 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
), 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 ). 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 () and 1A7 coated () 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
). 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
). 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
). 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
). 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.