With dose escalation up to 4 mg/m2
/day × 3 days per cycle, we identified an MTD of 3.0 mg/m2
/day. This dose is substantially lower than the MTD of 6.4 mg/m2
/day determined in a previous phase 1 trial of huKS-IL2 as single-agent therapy in patients with prostate cancer [19
]. In that study, 4 of 22 patients experienced DLTs (dyspnea, hypotension, symptoms related to a vascular-leak syndrome: hypotension, chills, rigors, and crackling at the base of the lung; hypertension, anemia, and asthenia) [19
]. In the current study, DLTs were observed in 4 of 27 patients. At the highest dose level of 4.0 mg/m2
, dose-limiting myleosuppression occurred in 2 patients. Hypoxia, dyspnea, bronchospasm, and GGT increase were also observed. However, we could not identify a consistent pattern of toxicity, and typical signs of a systemic vascular-leak syndrome were not detected. Profound (≥ grade 3) non-dose-limiting lymphocytopenia was observed in 5 patients (at a dose ≥ 2.0 mg/m2
in 4 patients). Consistent with other IL2/cytokine-based therapies, these episodes of lymphopenia may not reflect myelosuppression, but are likely due to immunocytokine-induced lymphocyte trafficking to the peripheral tissues [20
], including tumor beds or draining nodal tissues.
The estimated huKS-IL2 Cmax
values varied widely within and between huKS-IL2 dosing cohorts, within cohort coefficients of variation ranging from 8.9% to 52.6% for Cmax
and from 16.5% to 106.1% for AUC0–24h
. A dose-dependent, but not dose-proportional, increase in Cmax
was observed for huKS-IL2 over the tested dose range. Cmax
was achieved within 1 hour after the end of the 4-hour infusion period. No clinically relevant accumulation of huKS-IL2 was observed either within (ratio for AUC0–24h
ranging from 0.69 to 1.37) or between (ratio for AUC0–24h
ranging from 0.55 to 1.23) treatment cycles. Terminal half-life and systemic clearance did not tend to change with dose or over time. These PK results are consistent with those obtained in previous clinical studies [19
], in which Cmax
was also achieved within 1 hour after infusion end and displayed a dose-dependent, but not dose-proportional, increase in concentrations and exposure.
There appear to be some similarities in the PK activity of immunocytokines. Similarities between huKS-IL2 and published PK data for hu14.18-IL2 (EMD 273063), a humanized anti-GD2 monoclonal antibody linked to IL2, included that Cmax
was achieved at the end of the 4-hour infusion period, increases in Cmax
and AUC were dose dependent, and there was no accumulation of the immunocytokines [21
]. However, in contrast to huKS-IL2, a dose-dependent decrease in clearance was observed with hu14.18-IL2, and both clearance and mean peak concentrations increased in cycle 2 [22
]. These differences may be related to the specific target antigens.
Most patients treated with huKS-IL2 had a transient immune response, commonly anti-idiotype or anti-linker (the engineered component that links the IL2 to the antibody) antibody induction. Whether or not these antibody responses have any detrimental/neutralizing effects on huKS-IL2 is not known. As in other antibody-based therapies, it is also possible that the induction of anti-idiotype antibodies could enhance the anti-EpCAM effects via downstream antibody responses. In a monotherapy phase 1 trial, immunologic activity of huKS-IL2 was demonstrated by increases in lymphocyte counts, NK cell activity, and antibody-dependent cellular cytotoxicity [19
]. In the current study, we monitored immune response to therapy by serial flow cytometric evaluation of PBMC populations (T-cell subsets, NK cells, and T-reg cells), serum-soluble IL2 receptor determination, and PBMC response to tetanus toxoid. Consistent with the effects on immune function of exogenous IL2, PBMC counts decreased during the days of huKS-IL2 infusion, reflecting treatment-related lymphopenia/lymphocyte trafficking. Rebound of PBMC populations to baseline levels, and often higher, was observed within a week of the end of infusion, with rebound appearing to be more pronounced at higher huKS-IL2 doses. In contrast, there was no evidence of dose-dependent huKS-IL2 effects on the increase in sIL2R levels observed in vivo
or the PBMC response to tetanus toxoid in vitro
The rationale for combining huKS-IL2 with low-dose cyclophosphamide in the present study was based on the earlier observation that certain cytotoxic chemotherapeutic agents sensitize tumors to the tumoricidal effects of the immunocytokine within the tumor microenvironment [18
]. Sensitization may occur as a result of reducing intra-tumoral pressure and lowering the macromolecular diffusion barrier, thus potentially permitting increased uptake of the immunocytokine antibodies. Moreover, existing clinical evidence suggests the combination of low-dose cyclophosphamide together with a continuous-infusion or IL2 or other related biologics yields significant anti-tumor activity in patients with advanced cancers [23
]. These effects have been shown to be related to differential loss of regulatory T-cells compared with cytotoxic lymphocyte populations.
In a study that evaluated the cancer vaccine IMA901 in patients with renal cell carcinoma, pre-treatment with a single dose of cyclophosphamide was shown to down-modulate T-regs and contribute to overall survival [25
]. Patients receiving cyclophosphamide had a significant decrease of median T-reg levels 3 days after cyclophosphamide treatment. In the current study we cannot specifically comment on the status of T-reg cells in response to the cyclophosphamide alone, as the addition of huKS-IL2 on days 2–4 resulted in loss of T-reg cells followed by rebound, as would be expected with IL2 effect. However, a previous study that evaluated recombinant IL2 (rIL2) and low-dose cyclophosphamide in patients with malignant melanoma and renal cell carcinoma reported that the regimen had minimal anti-tumor activity—no remission in renal cell carcinoma patients and partial or minor response in 3 (17%) melanoma patients [26
]. An additional 4 patients had stable disease (1 with renal cell carcinoma and 3 with melanoma).
Based on pre-clinical ex vivo
studies and animal models, it is likely that immunocytokines have clinical effects by both NK cell- and T-cell-mediated mechanisms [27
]. The immunocytokines facilitate antibody-dependent cellular cytotoxicity (ADCC) in vitro
with both healthy donor effector cells and with immune cells from patients with advanced cancer. Similarly, depletion of NK cells dampens the anti-tumor activity of immunocytokines in mouse models. Although NK cells play a role in immunocytokine therapy, pre-clinical data also support the key role of T-cell-mediated effects, including the development of memory that is protective against subsequent tumor challenge. Therefore, it is theorized that immunocytokine therapeutic effects result from NK-mediated ADCC of tumor cells that contributes to direct tumor cell death, and that this effect results in release of antigens that can facilitate/potentiate the development of anti-tumor T-cell populations. These T-cells can then act as primary anti-tumor effectors as well as providing long term memory effects. The effects on T-reg cells and the potential benefit in terms of induction of T-cell immunity was one of the main rationales for the use of cyclophosphamide in this study.
With regard to anti-tumor activity in the current study, no objective responses were observed. Ten patients had stable disease as best response and 7 patients were alive at the time of analysis. As this was a phase 1 study, the primary objective was not to evaluate response to therapy. The clinical utility of immunotherapy in general has been best seen in the setting of minimal residual or sub-clinical disease. For this reason it was not expected that huKS-IL2 therapy (alone or with low-dose cyclophosphamide) would demonstrate clinical responses in patients with end stage clinically measurable (and often bulky) disease. Although not studied as a specific endpoint, several subjects have had excellent responses to additional cytotoxic therapies and have become long-term survivors. Granted, the number of cases is very small; however, no pattern or distinctive features such as pre-treatment lymphocyte patterns are seen in these subjects. At this time continued follow-up on this handful of cases is of particular interest.
For these reasons, one could argue that both the higher-dose monotherapy and the combination of cyclophosphamide with lower doses of huKS-IL2 should/could be developed further. A potential next step that could address these issues would be to move to a randomized phase 2 study of both regimens, with anti-tumor effect as the primary outcome. Clearly a study such as this would require close monitoring and early stopping strategies in case one regime is found to be more effective.