Transient T cell depletion causes regression of melanoma metastases

Of the common adult-onset cancers, melanoma is widely held to be the most amenable to immunotherapeutic intervention. This perception is based on the following: (i) several melanoma-specific antigens have been identified; (ii) melanoma antigen-specific CD4⁺ and CD8⁺ T lymphocytes are increased in melanoma patients and have anti-tumor activity; (iii) immune-enhancing agents can cure mice of established melanomas; and (iv) spontaneous melanoma regressions in humans with the simultaneous onset of vitiligo have been reported [1, 2]. Additionally, the immune-enhancing agent, interferon-α, increases survival in melanoma patients with intermediate or high risk of recurrence, and high-dose interleukin 2 (IL-2), a potent stimulator of T cell proliferation, causes durable remissions in a small subset of patients with metastatic melanoma [3–5].

Cognate immune reactions against tumor cells depend on a balance between activated tumor antigen specific CD4⁺ and CD8⁺ T cells and suppressive regulatory T cells [6]. CD4⁺CD25^HI regulatory T (Treg) cells directly suppress the activation of anti-tumor effector T cells in a contact-dependent manner [7, 8]. Depletion of these immunosuppressive Treg cells using anti-CD25 monoclonal antibodies enables a CD8⁺ and CD4⁺ T cell dependent immune rejection against the progression of melanoma in mice [7, 9, 10]. This anti-tumor activity is not exclusive to melanoma since it has been observed against several transplantable mouse tumor cell lines in multiple disparate mouse strains [11–13]. Furthermore, down-regulation of Treg cell function can be accomplished with certain stimulatory antibodies (e.g. anti-GITR [DTA-1]) and this treatment confers concomitant tumor immunity against remote tumors even in poorly immunogenic mouse melanomas [10]. Treg cells thus may function to tolerize or deactivate cognate effector CD4⁺ and CD8⁺ T cells to neoplastic cells that express tumorigenic antigens. Accordingly, the selective depletion of these cells may have clinical utility for the induction of anti-tumor immunity.

A multitude of successful treatment strategies in tumor-bearing mice have failed when examined in human clinical trials, and correlative data that provide additional rationale for clinical trials in humans is essential. Viguier et al. reported the results of an immunohistochemical examination for the presence of CD4⁺CD25^HI Treg cells in lymph nodes with and without metastasic melanoma seeds extracted from twelve patients with metastatic melanoma (Stage IIIb/IIIc) [14]. They observed that the frequency of Treg cells in melanoma-positive lymph nodes was 11.06% whereas the frequency was only 6.2% in melanoma-free lymph nodes [14]. The over-representation of Treg cells in metastatic lymph nodes suggests that these cells may be recruited and/or expanded at the tumor site. Treg cells isolated from the metastatic lymph nodes were found to inhibit CD4⁺ and CD8⁺ T cell proliferation in vitro via a contact-dependent mechanism and thus appear to function similarly to the well characterized mouse Treg cells. Taken together, these data provide correlative evidence that CD25⁺ Treg cells may be functioning to suppress anti-tumor immunity in melanoma patients.

Denileukin diftitox (DAB/IL2; Ontak) is a recombinant DNA-derived cytotoxic protein composed of diphtheria toxin fragments A and B and the full-length IL-2 molecule. DAB/IL2 binds to CD25 (the α chain of the IL-2 receptor) and, following internalization, inhibits protein synthesis, causing cell death within hours [15]. DAB/IL2 is FDA-approved for the treatment of patients with persistent or recurrent cutaneous T cell lymphoma (CTCL) whose malignant cells express CD25. CTCL malignant proliferation is driven by interaction with cutaneous dendritic cells (termed Langerhans cells), and this epidermal association of CTCL cells with Langerhans cells is a hallmark of the malignancy [16]. In recent studies, purified human CTCL cells were incubated with dendritic cells and found to adopt a Treg phenotype, including: (i) expression of the negative T cell regulator, cytotoxic T lymphocyte antigen-4 (CTLA-4); (ii) expression of the Treg specific transcription factor, Foxp3; and (iii) the ability to suppress CD4⁺ T cell activation [16]. Based on these findings, CTCL has been postulated to be a malignant proliferation of Treg cells and, coupled to the observation that Treg cells express high surface CD25, these data provide rationale for the testing of DAB/IL2 as a selective Treg-depleting agent.

In the current study, we have found that DAB/IL2 administration to stage IV melanoma patients depletes peripheral blood Treg cells and causes the regression of metastatic tumors in a subset of patients. We also observed a significant depletion of the total T cell population including CD4⁺CD25^- T cells that was reversed within 21 days of DAB/IL2 administration. We speculate that transient depletion of T cells in cancer patients may disrupt the homeostatic control of cognate immunity and allow an expansion of CD8⁺ T cells with increased specificity for the peptide/MHC complexes expressed by neoplastic cells.

Melanoma incidence has risen by 25–31% over the last decade and is now the 5^th most common cancer in men and the 6^th most common cancer in women [1, 17]. Melanoma causes a disproportionate mortality in young and middle-aged individuals and, as such, displays one of the highest "loss of potential life" rates among the adult-onset cancers (18.6 years per melanoma-related death) [1]. In the United States, over 8000 adults die of melanoma annually, and 84% of melanoma patients with distant metastases succumb to their disease within 5 years of diagnosis [1].

The treatment options for patients with metastatic melanoma are limited to palliation or to aggressive therapy with high dose IL-2 or biochemotherapy using cisplatin, vinblastine, dacarbazine, IL-2 and interferon α-2b. The response rate to high dose IL-2 is low (16%) but durable cures have been observed in approximately 6–10% of the patients that can tolerate the systemic toxicity (i.e. hypotension, capillary leak syndrome, sepsis and renal failure) [3, 4]. Although biochemotherapy has been reported to yield a 35–50% partial response rate and up to a 20% complete response rate, median survival duration is only 12.2 months [17, 18]. Early published reports of clinical trials of humanized anti-CTLA4 monoclonal antibodies have indicated a 10–20% partial response rate in melanoma patients [19]. In the current study, we observed a 31% partial response rate after treatment with DAB/IL2 (5/16 patients) which is clinically significant given the low toxicity of this agent. Importantly, the majority of patients who are treated with high dose IL-2, biochemotherapy and/or anti-CTLA4 ultimately experience progression and few efficacious alternative treatments are currently available.

We found that transient depletion of CD4⁺ and CD8⁺ T cells in melanoma patients via targeting of IL-2 receptor-expressing cells resulted in T cell repopulation in the peripheral blood and the de novo appearance of CD8⁺ T cells with specificity for melanoma antigens (in 4/7 HLA-A2*0201 patients). We had anticipated that the detection of peripheral blood MART1-, gp100- and tyrosinase-specific CD8⁺ T cells in these HLA-A2*0201⁺ patients might correlate with tumor regressions. The three HLA-A2*0201⁺ patients who did not develop any detectable MART1-, gp100- and tyrosinase-specific CD8⁺ T cells also did not experience regression of their melanoma metastases (Table 1). However, we observed the regression of melanoma metastases in only 2/4 HLA-A2*0201⁺ patients who developed melanoma antigen-specific CD8⁺ T cells (Table 1). We can only speculate that the two patients who developed melanoma antigen-specific CD8⁺ T cells but did not experience tumor regressions may have melanomas that express low class I MHC or effector CD8⁺ T cells that are compromised by low affinity T cell receptors and/or the tumor microenvironment. Importantly, the peptide/MHC tetramers used in this study can only detect a miniscule fraction of the possible CD8⁺ T cells that have specificity for MART1, gp100, tyrosinase or other melanoma antigens.

Intriguingly, patients P3 and P14 experienced the regression of multiple metastastic melanomas simultaneously with the persistence and even growth of other metastatic melanomas (i.e. a mixed response). The residual peri-aortic mass in patient P14 was confirmed to express the melanoma antigen, MART1, and this patient developed peripheral blood MART1-specific CD8⁺ T cells within 21 days of transient T cell depletion. Despite immunohistochemical evidence that CD8⁺ T cells appeared to surround the MART1⁺ melanoma cells, this residual metastatic melanoma was not cleared. Interestingly, the melanoma cells did not express the class I MHC proteins HLA-A, B or C which may partly explain the lack of regression of this particular metastasis. We suspect that differences in melanoma antigen expression and/or additional tumor immunoevasion tactics also may explain such differential anti-tumor effects within a single host and future studies will be directed at further examination of the phenotypes of melanoma cells and infiltrating immune cells in growing and regressing melanomas within a single host.

DAB/IL2 administration transiently decreased CD4⁺CD25^-, CD4⁺CD25⁺, CD4⁺CD25^HIFoxp3^-, CD4⁺CD25^HIFoxp3⁺, CD8⁺ T cells and, in certain patients, melanoma antigen-specific CD8⁺ T cells. These data suggest that DAB/IL2 is not selectively cytotoxic to T regulatory cells which may be due, in part, to the high IL-2 receptor expression of activated effector T cells. We found that all examined T cell subsets repopulated the peripheral blood and presume that this repopulation is due either to a proliferative expansion or re-trafficking of T cells from lymph nodes. Interestingly, CD4⁺ or CD8⁺ T cell depletion in mice has been found to cause a proliferative expansion of the residual T cells that restores the original T cell pool size [20]. This peripheral expansion has been termed homeostatic proliferation and can prevent the induction of tolerance to transplanted organs and cause anti-tumor responses against melanomas and colon cancer in mice [20–22]. Although the mechanisms for these effects are not well established, homeostatic proliferation of CD4⁺ and CD8⁺ T cells is, in part, driven by MHC/peptide recognition. We postulate that transient T cell depletion in cancer patients may cause a rebound expansion of T cells with a shifted TCR repertoire that includes increased melanoma antigen-specific CD8⁺ T cells. On-going studies that examine the relative effects of Treg depletion and transient total T cell depletion on melanomas in mice and the consequences of adding back Treg cells or total lymphocytes on these effects should improve our understanding of the precise mechanisms for the observed tumor regressions in melanoma patients.

The effect of DAB/IL2 on both peripheral blood Treg cell concentration and tumor burden has been previously examined in 4 patients with metastatic breast, lung or ovarian cancer (single infusion; 9 μg/kg or 12 μg/kg) [23]. Prior to infusion, the mean Treg cells/mm³ was found to be 126 (26.8% of total CD4+ Tcells) and, after DAB/IL2 infusion, the mean Treg cells/mm³ was reduced to 78 (19.0% of total CD4+ Tcells) [23]. A patient with Stage IIIC, relapsed ovarian cancer received the highest dose of DAB/IL2 (12 μg/kg) and experienced a marked reduction in the ovarian cancer marker, CA-125, four weeks after a single infusion (from 121 U/ml to 38 U/ml) [23]. This patient was then administered 6 additional doses of DAB/IL2 weekly (12 μg/kg) and, two months after the final dose, a PET/CT fusion scan revealed a dramatic reduction in metastatic burden. In a second study, twelve stage IV melanoma patients were administered DAB/IL2 at a lower dose (9 μg/kg) and a higher dose (18 μg/kg) daily × 5 days every three weeks [24]. The investigators reported that none of the patients experienced an objective clinical response and that the expression of the Treg-specific transcription factor Foxp3 and the suppressive ability of CD4⁺CD25^HI cells did not decrease significantly. The disparity in clinical results between this study and the current study of melanoma patients may be due to differences in: (i) the dosing (9 or 18 versus 12 μg/kg); (ii) the duration of treatment in each cycle (5 daily versus 4 daily doses per cycle); (iii) the low sample size in both studies; (iv) the extent of disease in the examined human subjects; (v) the methods used to measure T regulatory cells (real-time PCR for Foxp3 versus three color flow cytometry for CD4⁺CD25^HIFoxp3⁺ cells); and (vi) the extent of prior immunotherapeutic treatments that had been administered to the patients in each study (e.g. IL-2). Importantly, in a recent study, DAB/IL2 was found to significantly reduce the peripheral blood Treg cells in metastatic renal cell carcinoma patients and to abrogate Treg-mediated immunosuppressive activity in vivo [25]. This study also demonstrated that DAB/IL2-mediated depletion of Tregs followed by vaccination with RNA-transfected DCs increased the activation of tumor-specific T cell responses compared to vaccination alone [25].

In conclusion, we have demonstrated that T cell depletion with DAB/IL2 in melanoma patients is followed by a T cell repopulation of the peripheral blood, the de novo appearance of CD8⁺ T cells specific for melanocyte differentiation antigens and regression of melanoma metastases. This single site, investigator-initiated phase II clinical trial has been expanded to provide further evidence for the efficacy of DAB/IL2 against melanoma. Future clinical studies that are conceptually appealing include an examination of the efficacy of DAB/IL2 in several cancer types and the potential utility of combination therapy with vaccines or other immune enhancing agents such as interferon-α. Last, we anticipate that the limited or pulsed administration of alternative T cell depleting agents may prove useful for the activation of cognate immunity against neoplastic cells in cancer patients.