NK-mediated lysis of neuroblastoma cell lines in vitro
is augmented by the addition of IL2 (20
), especially when using NK cells from patients receiving IL2 (21
). Furthermore, NK-mediated antibody dependent cell-mediated cytotoxicity (ADCC) is augmented when the NK cells are obtained following in vivo
administration of IL2 (22
). Initial studies with chimeric anti-GD2 antibody, ch14.18, fused with human IL-2 (ch14.18-IL2), demonstrated NK-mediated regression of local and disseminated murine neuroblastoma (15
). Similar results were later seen in murine neuroblastoma models with administration of hu14.18-IL2(16
). As escape from this NK-mediated response to hu14.18-IL2 was associated with up-regulation of MHC-class I on NBL cells (known to induce inhibitory responses via Ly-49 receptors on murine NK cells) (17
), we hypothesized that similar relationships may influence the clinical response to hu14.18-IL2. In order to test this hypothesis, it was first necessary to identify a population that shows some clinical response to hu14.18-IL2.
Our recently reported Phase II study of hu14.18-IL2 in patients with relapsed or refractory NBL demonstrated CR or improved disease in 7 of 38 treated patients(18
). All 7 of these responding/improved patients were in Stratum 2 (evaluable but not radiographically measurable disease), consistent with our preclinical data showing greater detection of anti-tumor activity in animals with less tumor burden (16
). Although the role of KIRs has been evaluated in the setting of autologous and allogeneic stem cell transplantation and infusions of allogeneic NK cells following lymphodepletive chemotherapy (1
), it has not been studied for association with antitumor response in patients receiving only cytokines or monoclonal antibodies for immunotherapy. We hypothesized that children with recurrent/refractory NBL who received the hu14.18-IL2 in our Phase II COG study would demonstrate greater response to IC in the presence of KIR/KIR -ligand mismatch. When the data were analyzed for all 38 patients that provided DNA samples, 24/38 patients were found to be KIR/KIR -ligand mismatched, and all 7 of the responding/improved patients were found in this group (p = 0.03, ). Since the KIR/KIR-ligand interaction is primarily a mechanism controlling NK cell activity, this result is consistent with the murine data showing that the anti-neuroblastoma effect of ch14.18-IL2 and hu14.18-IL2 is primarily mediated by NK cells (15
). Even prior to the administration of hu14.18-IL2, there is a trend towards greater KIR/KIR-ligand mismatch in those patients that enter Stratum 2 than Stratum 1 (p = 0.08, ). If additional data validate this trend, it would suggest that a child’s endogenous KIR/KIR-ligand status may play a role in the clinical pattern of relapse; namely, those patients that are KIR-mismatched may be less likely to relapse with “bulky” (measurable/Stratum 1) disease. Similarly, if additional data validate the trend (p = 0.13) that KIR/KIR-ligand mismatch is associated with response within stratum-2 patients (), the presence of stratum-2 status and KIR/KIR-ligand mismatch might be considered as eligibility criteria for future treatment with hu14.18-IL2 for children with relapsed or refractory neuroblastoma.
The roles of the activating Fc receptors involved in ADCC, FcγR2A and FcγR3A, have been demonstrated in response to rituximab, cetuximab and trastuzumab (7
). Cheung et al have found an association between FcγR2A polymorphism and outcome of neuroblastoma patients receiving the murine anti-GD2 IgG3 antibody, 3F8, but only when given in combination with GM-CSF. This result suggests that when neutrophils or monocytes/macrophages are activated with GM-CSF, they facilitate clinically meaningful ADCC via the high affinity alleles of FcγR2A for 3F8(12
). This response was unlikely to be NK-mediated, as FcγR2A is not present on NK cells. As preclinical data and our current results () suggest that NK cells are playing a role in the clinical response to hu14.18-IL2 in NB patients, we initially hypothesized that the response/improvement of neuroblastoma patients to the IgG1-containing IC might be associated with the presence of the high-affinity FcγR3A 158-V/V genotype (influencing NK function), and not the high-affinity FcγR2A 131-H/H genotype reflecting neutrophil and macrophage ADCC. However, we found that patients with the H/H genotype for FcγR2A did show a trend toward higher response rate, which was of marginal statistical significance (p = 0.06). Clearly these comparisons are underpowered. Even so, the near significant association of improvement with the FcγR2A 131-H/H genotype would be consistent with the activation of some neutrophil or macrophage-mediated ADCC. IL2 treatment is known to induce release of GM-CSF by IL2 responsive cells (23
), and it is possible that activation of ADCC by these FcγR2A-bearing cells is resulting from GM-CSF production stimulated by the IL2 component of the IC.
Given the infrequency of the FcγR3A 158-V/V genotype in the general population, with only 2 of our 36 genotyped patients identified with this genotype, it is no surprise that a statistically significant association was not seen between this genotype and response. However, this lack of statistical significance cannot be equated with the lack of proof in this study due to inadequate sample size with a highly unbalanced design. Even so, it may be surprising that none of the 7 patients that showed response or improvement had this V/V genotype. This very preliminary result would suggest that there may not be an association between the V/V genotype and improvement in this population when treated with hu14.18-IL2. The lack of an association with FcγR3A alleles and response (even if confirmed in a larger sample) does not necessarily indicate that NK cells are uninvolved in mediating ADCC. Rather, it could suggest that there is no advantage for high vs. lower affinity FcγR3A alleles in this setting of IC-mediated ADCC. We have recently shown that some NK cells can use their IL2 receptors to recognize the membrane-bound IL2 on tumor cells coated with tumor-reactive IC (Gubbels et al submitted). This results in NK adhesion to the IC-coated tumor cells, activation of an immune synapse and subsequent tumor cell destruction, apparently without requiring Fc receptors (Buhtoiarov et al, submitted). In the face of this IL2 receptor-facilitated IC-mediated killing, high affinity FcγRs on the NK cells might not be needed. In other words, while high affinity FcγRs play an important role in the clinical effects of FcR-mediated ADCC using conventional mAbs, they might not be as important for NK mediated antitumor effects of ICs. This might enable NK cells with the F/V or F/F FcγR3A alleles to mediate comparable in vivo destruction to that mediated by cells with V/V alleles. Clearly, these are speculative hypotheses as the results were obtained from a limited number of subjects and must be extended to a larger population to draw firmer conclusions.
The COG plans to perform a follow-up study of hu14.18-IL2 in Stratum 2 patients, to further characterize these responses. That follow-up study should provide additional data to better test the associations analyzed in this report. Furthermore, the KIR/KIR-ligand mismatch might also be further tested by genotyping of subjects participating in larger trials of clinically effective mAbs that mediate ADCC, such as ch14.18, rituximab, cetuximab and trastuzumab(7
In summary, we conclude that response or improvement of relapsed or refractory neuroblastoma patients following treatment with hu14.18-IL2 is associated with KIR receptor-ligand mismatch, consistent with a role for NK cells in this clinical response. While there appears to be no statistical significance of FcγR genotype in response of neuroblastoma patients to IC in this COG trial, further clinical and in vitro data are needed to better clarify the potential roles of FcγR2A and FcγR3A in these clinical responses.