To investigate whether the amount of induced anti-IGN- 311 antibodies is, at least theoretically, sufficient for binding the LeY antigen, the IGN311 serum concentration during the treatment course was determined (Figure 4). Although IGN311 has shown a half-life in patient serum  of 10 - 12 days after the first infusion and of ~24 days following the second infusion of 100 mg IGN-311/ patient , which is close to the reported t1/2 of 21 days of natural IgG, patient 1 showed a rather different picture.
Whereas after the 1st infusion, the pharmacokinetics (measured until day 8) were similar to the other two patients, a dramatic difference was evident 2 days after the 2nd infusion: in contrast to patient 2 whose IGN311 titer slowly decreased according to expected serum half-life, no IGN311 was detectable in patient 1. Based on the presented data so far, the explanation for this observation is that IGN311 can form complexes with the idiotypespecific antibodies generated by the HAHA response and can therefore not be detected by ELISA based on an antiid antibody used for detection. In accordance with the rapid complex formation of IGN311 with the idiotype specific antibodies also the effector function are abrogated rapidly. To get information about how much anti-id antibodies were produced in patient 1, we used the IGN- 311 anti-id antibody MMA383 as calibrator to quantitate the HAHA response (Table 1).
At peak levels (at day 15) around 15 µg/ml MMA383 equivalents are present in serum which are then steadily decreasing to approximately 6 µg/ml at day 65. In contrast, a ten-fold lower amount was detected at day 8 indicating that this amount of HAHA response is most likely not sufficient to neutralize IGN311 (assuming a 1:1 stoichiometry). The measured amount of unbound IGN311 (Figure 4) correlated with the decrease in CDC and ADCC activity measured in the serum of patient 1 (Figures 3(a) and (b)).
3.4. Ab3 Induction
Regarding the correlation of the HAHA response with
Table 1. HAHA quantification. Using the murine mAb MMA383 as reference which recognizes the idiotype of IGN311 in a qualified BIACORE assay  the RU’s were assigned to an amount of MMA383 equivalents (µg/ml).
Figure 4. Determination of IGN311 levels in patient sera (ELISA). MMA383 was used to capture IGN311 present in patient serum at time points (hours) 0 (day 1, 1st infusion), 0.5, 4, 8, 24 (day 2), 48 (day 3), 96 (day 5), 168 (day 8), 336 (day 15, 2nd infusion), 336.5, 340, 344, 360 (day 16), 384 (day 17), 432 (day 19), 504 (day 22), 672 (day 29), and 1008 (day 43). Patient 1 (diamonds), patient 2 (circles).
the disease stabilization seen in patient 1, we hypothesized that the induction of the anti-idiotypic HAHA (Ab2) response against IGN311 (Ab1) may consequently result in generation of anti-anti-idiotypic antibodies (Ab3) [19, 20] which recognize LeY on the target cells leading to an in situ immunization against the per se non-immu-nogenic carbohydrate LeY target antigen . Therefore, we used an ELISA approach to verify induction of Ab3 antibodies. False positive signals resulting from remaining serum IGN311 (IgG1/κ) were eliminated by an ELISA detection system specific for the human λ chain. In humans, the ratio of antibodies containing κ or λ light chains is 60:40  therefore the chance for the existence of a specific Ab3 clone expressing a λ light chain or λ light chain is roughly equal. As can be seen in Figure 5, patient 1 developed a significant λ chain related anti-id (antiMMA383) reactivity.
The induction of an immune response against passively administered murine therapeutic antibodies (HAMA) has been frequently reported and was found to be associated with severe side effects and unfavorable pharmacokinetics. On the other hand, HAMA can also be associated with a positive clinical outcome  because—apart from direct cytotoxic mechanisms such as ADCC, CDC and induction of apoptosis—Ab2 and Ab3 have been postulated according to the network hypothesis of Jerne to induce tumour cell rejection [3,23]. According to Jerne’s network theory, a subset of anti-idiotypic Abs (Ab2beta)
carries an “internal image” of the Ag and induces Abs (Ab3) against the original Ag [Jerne, 1974]. Whereas an immune response against murine Abs can be directed against all parts of the molecule which are non-homologous to their human counterparts, humanized mAbs should only be immunogenic with regard to the murine amino acid sequences in their antigen binding site. Consequently, a human anti-human antibody (HAHA) response should be idiotype-specific. Interestingly, investigations regarding the induced HAHA responses in clinical trials revealed that application of different humanized antibodies (although all IgG1/κ) induced different levels of HAHA . Whereas passive application of Herceptin or Avastin induced marginal levels of HAHA [24,25], application of huA33  resulted in greater than 50% HAHA responders indicating that a) humanization does not resolve all immunogenicity issues and that b) the occurrence of HAHA response cannot be predicted. The high incidence of HAHA induction by a humanized antibody huA33 was also shown by the data obtained with IGN311. Additionally, the high frequency of HAHA was in all three cases measured by a surface plasmon resonance approach and not by ELISA. This finding warranted a side-by-side comparison of both techniques to assess the possibility of identifying false positive/false negative HAHA responders. Such approach was taken by Lofgren et al.  and indeed they identified more HAHA positives samples when applying SPR technology.
Regarding an anti-anti-idiotypic response, Cheung et
Figure 5. Determination of Ab3 antibodies after IGN311 (Ab1) application (ELISA). MMA383 was used to capture Ab3 antibodies in patient serum. Sera were diluted 1:10 and bound antibodies were detected with a human λ light chain specific HRP conjugate. Values shown have been blanked against day 0. For graphical depiction, day 0 was set to an absorbance value of 0.05. n.d = not determined.
al.  showed that Ab3 antibodies are induced after application of the anti-GD2 mAb 3F8. Furthermore, Ab3 induction was associated with long term survival supporting the network concept postulated by Jerne. The finding that high HAMA titers, resulting in IgG Ab3 antibodies, prolong survival, are consistent with the finding that IGN311 treated patient 1 showed stable disease over the whole treatment period and post-treatment observation period. Uttenreuther-Fischer et al.  analyzed the details of the immune response in a neuroblastoma patient also with molecular methods and found that the idiotypic Ab (Ab1) against the target GD2, a disialoganglioside overexpressed in neuroblastoma cells triggered the production of an anti-idiotypic Ab (Ab2). This Ab2 belonged to the Ab2beta subclass, which means that it mirrors the spatial structure of the Ag recognized by the therapeutic antibody (Ab1). Most importantly, this Ab2beta was capable of inducing the production of an anti-anti-idiotypic antibody, designated Ab3. This Ab3 in turn was able to bind the original tumor-associated antigen, resulting in a secondary immune response against the tumor cells. According to the authors, Ab3 production started with a delay of 6 - 14 months relative to the first application of the initial therapeutic mAb but then the production persisted for years, significantly contributing to the long-lasting, positive effect of the Ab therapy. Additionally to its beneficial effect in the patient, Ab3 could also be used as a surrogate marker for the success of the immune-therapeutic intervention.
The present data show that patient 1 receiving IGN311 (lowest dose, 50 mg/kg) developed a λ chain related anti-idiotypic (anti-MMA383) immune response. The observed long-term benefit from the immune therapy was presumably also, at least partly, mediated by the mechanisms originally described by Uttenreuther-Fischer et al. This is also supported by the finding that the blood-levels of Ab1 in patient 1 decreased very quickly after the second Ab injection, a hint that production of the antiidiotypic antibody resulted in a fast clearance of the therapeutic antibody. The rapid clearance of the antibody might also explain the reduced ability of the serum of patient 1 to induce immunogenic effects in the CDC and ADCC assays. Importantly, Ab3 is supposed to directly recognize LeY modified epitopes on the targeted tumor cells, thereby triggering additional effector functions in situ and even possibly facilitates the induction of an active immune response that specifically attacks these malignant cells for long term. This proposed effect presumably also contributes to the improved long-term outcome of IGN311 therapy seen in patient 1, despite a clearly compromised CDC and ADCC reactivity measured in the serum from this patient shortly after application of the therapeutic antibody. The induction of the anti-idiotypic responses following passive application of therapeutic antibodies may induce an immunization against the original antigen, by mimicking the original antigen by a part of the anti-idiotypic antibodies. Such active immunization component may prolong significantly the therapeutic effects triggered by the passive application of therapeutic antibodies. Moreover, in particular also for low immunogenic antigens such as carbohydrate antigens (including LeY) which are not able to induce an efficient immune response by themselves the mimicking image presentation by the anti-idiotypic antibodies can significantly enhance the induction of effective immune responses against the original antigen. Furthermore, it was also shown that the binding of IGN311 to LeY-modified receptors induced an inhibition of signal transduction via these receptors . This mechanism additionally enhanced the effect of the immunotherapy because co-expression of LeY antigen and of EpCAM receptors results in a poor prognosis for these patients .
The presented data shed some light on the immunological basis for the long-term clinical disease stabilization found in one patient who developed a pronounced HAHA type I response after infusion of the humanized antibody IGN311. This also encourages further research to clarify the exact molecular mechanisms underlying the described effects. In the light of the recently published literature, this might help to further improve the study design and implementation of future clinical trials, resulting in a less burdening treatment and a positive longterm outcome in treated patients by inducing an active immunization component following passive application of therapeutic antibodies .
A. N. and R. K. thank Dr. Gerald Schmid for manuscript review.