Trine Zeeberg Iversen

Adoptive cell therapy with autologous tumor infiltrating lymphocytes and low-dose Interleukin-2 in metastatic melanoma patients

BackgroundAdoptive cell therapy may be based on isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL), in vitro activation and expansion and the reinfusion of these cells into patients upon chemotherapy induced lymphodepletion. Together with high-dose interleukin (IL)-2 this treatment has been given to patients with advanced malignant melanoma and impressive response rates but also significant IL-2 associated toxicity have been observed. Here we present data from a feasibility study at a Danish Translational Research Center using TIL adoptive transfer in combination with low-dose subcutaneous IL-2 injections.MethodsThis is a pilot trial (ClinicalTrials.gov identifier: NCT00937625) including patients with metastatic melanoma, PS ≤1, age <70, measurable and progressive disease and no involvement of the central nervous system. Six patients were treated with lymphodepleting chemotherapy, TIL infusion, and 14 days of subcutaneous low-dose IL-2 injections, 2 MIU/day.ResultsLow-dose IL-2 considerably decreased the treatment related toxicity with no grade 3–4 IL-2 related adverse events. Objective clinical responses were seen in 2 of 6 treated patients with ongoing complete responses (30+ and 10+ months), 2 patients had stable disease (4 and 5 months) and 2 patients progressed shortly after treatment. Tumor-reactivity of the infused cells and peripheral lymphocytes before and after therapy were analyzed. Absolute number of tumor specific T cells in the infusion product tended to correlate with clinical response and also, an induction of peripheral tumor reactive T cells was observed for 1 patient in complete remission.ConclusionComplete and durable responses were induced after treatment with adoptive cell therapy in combination with low-dose IL-2 which significantly decreased toxicity of this therapy.

Long-Lasting Complete Responses in Patients with Metastatic Melanoma after Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes and an Attenuated IL2 Regimen.

Purpose: Adoptive cell transfer therapy (ACT) based on autologous tumor-infiltrating lymphocytes (TIL) has achieved impressive clinical results in several phase I and II trials performed outside of Europe. Although transient, the toxicities associated with high-dose (HD) bolus IL2 classically administered together with TILs are severe. To further scrutinize whether similar results can be achieved with lower doses of IL2, we have carried out a phase I/II trial of TIL transfer after classical lymphodepleting chemotherapy followed by an attenuated IL2 regimen. Experimental Design: Twenty-five patients with progressive treatment-refractory metastatic melanoma, good clinical performance, age < 70 years, and at least one resectable metastasis were eligible. TIL infusion was preceded by standard lymphodepleting chemotherapy and followed by attenuated doses of IL2 administered in an intravenous, continuous decrescendo regimen (ClinicalTrials.gov Identifier: NCT00937625). Results: Classical IL2-related toxicities were observed but patients were manageable in a general oncology ward without the need for intervention from the intensive care unit. RECIST 1.0 evaluation displayed three complete responses and seven partial responses (ORR 42%). Median overall survival was 21.8 months. Tumor regression was associated with a higher absolute number of infused tumor-reactive T cells. Moreover, induction and persistence of antimelanoma T-cell responses in the peripheral blood was strongly correlated to clinical response to treatment. Conclusions: TIL-ACT with a reduced IL2 decrescendo regimen results in long-lasting complete responses in patients with treatment-refractory melanoma. Larger randomized trials are needed to elucidate whether clinical efficacy is comparable with TIL-ACT followed by HD bolus IL2. Clin Cancer Res; 22(15); 3734–45. ©2016 AACR.

Methods to Improve Adoptive T-Cell Therapy for Melanoma: IFN-γ Enhances Anticancer Responses of Cell Products for Infusion

Further development of adoptive T-cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has the potential to markedly change the long-term prognosis of patients with metastatic melanoma, and modifications of the original protocol that can improve its clinical efficacy are highly desirable. In this study, we demonstrated that a high in vitro tumor reactivity of infusion products was associated with clinical responses upon adoptive transfer. In addition, we systematically characterized the responses of a series of TIL products to relevant autologous short term-cultured melanoma cell lines from 12 patients. We provide evidence that antitumor reactivity of both CD8(+) and CD4(+) T cells could be enhanced in most TIL products by autologous melanoma sensitization by pretreatment with low-dose IFN-γ. IFN-γ selectively enhanced responses to tumor-associated antigens other than melanoma differentiation antigens. In addition, IFN-γ treatment was invariably associated with restored/increased cancer immunogenicity as demonstrated by upregulation of major histocompatibility complex molecules. These findings suggest a potential synergism between IFN-γ and ACT, and have important implications for clinical development of combination strategies for the treatment of metastatic melanoma.

Natural CD4+ T-Cell Responses against Indoleamine 2,3-Dioxygenase

Background The enzyme indoleamine 2,3-dioxygenase (IDO) contributes to immune tolerance in a variety of settings. In cancer IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it endorses the establishment of peripheral immune tolerance to tumor antigens. Recently, we described cytotoxic CD8+ T-cell reactivity towards IDO-derived peptides. Methods and Findings In the present study, we show that CD4+ helper T cells additionally spontaneously recognize IDO. Hence, we scrutinized the vicinity of the previously described HLA-A*0201-restricted IDO-epitope for CD4+ T-cell epitopes. We demonstrated the presence of naturally occurring IDO-specific CD4+ T cells in cancer patients and to a lesser extent in healthy donors by cytokine release ELISPOT. IDO-reactive CD4+ T cells released IFN-γ, TNF-α, as well as IL-17. We confirm HLA class II-restriction by the addition of HLA class II specific blocking antibodies. In addition, we detected a trend between class I- and class II-restricted IDO responses and detected an association between IDO-specific CD4+ T cells and CD8+ CMV-responses. Finally, we could detect IL-10 releasing IDO-reactive CD4+ T cells. Conclusion IDO is spontaneously recognized by HLA class II-restricted, CD4+ T cells in cancer patients and in healthy individuals. IDO-specific T cells may participate in immune-regulatory networks where the activation of pro-inflammatory IDO-specific CD4+ responses may well overcome or delay the immune suppressive actions of the IDO-protein, which are otherwise a consequence of the early expression of IDO in maturing antigen presenting cells. In contrast, IDO-specific regulatory T cells may enhance IDO-mediated immune suppression.

Immunological correlates of treatment and response in stage IV malignant melanoma patients treated with Ipilimumab

ABSTRACT Introduction: Ipilimumab is effective in the treatment of metastatic malignant melanoma, but few biomarkers reliably predict treatment response. Methods: Patients were treated with Ipilimumab for metastatic malignant melanoma. Blood and serum samples were collected before and during treatment. Mononuclear cells in peripheral blood were subjected to immune phenotypic analyses and cytokine levels were measured in serum samples. Results were correlated with clinical data. Results: A total of 40 patients were included in the analyses. Clinical response were associated with an increase after one series of treatment in absolute lymphocyte count (ALC) (p = 0.008), absolute T cell count (p = 0.02) and the absolute number of activated T cells in peripheral blood (p = 0.003). A high frequency of myeloid derived suppressor cells (MDSC) and a higher level of IL6 were associated with treatment failure, though not significantly. Levels of IL6 in serum above the median showed a tendency to associate with reduced survival by the 4th treatment series. Finally, treatment with Ipilimumab led to a decreased frequency of FOXP3+ regulatory T cells (p = 0.009). Conclusion: Ipilimumab leads to increased ALC, T cell count and T cell activation in malignant melanoma patients responding to treatment. A high baseline frequency of myeloid-derived suppressor cells and high levels of IL6 is associated with a reduced chance of responding to therapy.

Depletion of T lymphocytes is correlated with response to temozolomide in melanoma patients

Therapeutic strategies to deplete lymphocytes, especially regulatory T cells, in cancer patients have been proposed to increase the benefits of (immuno)chemotherapy. In this study, we explored the influence of temozolomide (TMZ) on different T-cell populations and addressed if the depletion of CD4+ T cells would be associated to the clinical benefits of TMZ. Patients were treated with TMZ (150 mg/m2 daily, every two weeks on a four-week schedule) until disease progression. Changes in T-lymphocyte subsets were characterized by flow cytometry. All patients enrolled in this study had histologically verified unresectable stage IV melanoma. Objective responses were induced in 12.5% of the patients, while 42.5% of them obtained short-term disease stabilization. The median progression-free survival (PFS) of this patient cohort was 8.7 mo. Lymphopenia (< 0.7 × 109 cells/L, grade 2) developed in 71% of the patients after 3 treatment cycles (~100 d). The development of grade 2 lymphopenia after the 3rd cycle of therapy positively correlated with clinical outcome (p = 0.01), and was linked, though non-significantly, to prolonged median PFS (303 vs. 200 d). In addition, significant changes in CD8+ T-cell subgroups were observed, notably a shift from naïve T cells toward more differentiated memory T cells. Finally, we demonstrated that specific CD8+ T-cell responses against selected tumor associated antigens (TAAs) were enhanced by the administration of TMZ (p = 0.04), while virus-specific T-cell responses were stable. Thus, immunological monitoring in the course of TMZ treatment might become an important tool for clinical guidance in the future.

Safety, immune and clinical responses in metastatic melanoma patients vaccinated with a long peptide derived from indoleamine 2,3-dioxygenase in combination with ipilimumab

BACKGROUND AIM Indoleamine 2,3-dioxygenase (IDO) is an emerging new target in cancer therapy that can be targeted with active immunotherapy (e.g. through peptide vaccination). Furthermore, IDO has been identified as a key mechanism underlying resistance to treatment with the checkpoint blocking antibody ipilimumab (ipi). METHODS Ten patients with metastatic melanoma participated in a phase I first-in-human clinical study assessing safety of combining ipi with a 21-mer synthetic peptide vaccine from IDO denoted IDOlong. Secondary and tertiary end points included vaccine and clinical response. RESULTS Treatment was generally safe and well tolerated. Vaccine related adverse reactions included grade I and II erythema, oedema and pruritus at the vaccination site, which were manageable with mild topical corticosteroids. One patient developed presumed ipi-induced colitis. It initially responded to high-dose parenteral corticosteroids but later relapsed while the patient was admitted to a local hospital, where he died after receiving suboptimal therapy. Vaccine-specific T-cell responses were detectable ex vivo in three patients. At first evaluation, five of the 10 treated patients were in stable disease, one of whom had an unconfirmed partial response. CONCLUSIONS Treatment with IDOlong synthetic peptide vaccine in combination with ipi was generally safe and without augmented toxicity. The vaccine induced readily detectable T-cell responses in a subset of patients. Treatment showed signs of clinical activity, although not exceeding efficacy of ipi alone. Results should be confirmed in a larger study.

mRNA-transfected dendritic cell vaccine in combination with metronomic cyclophosphamide as treatment for patients with advanced malignant melanoma

ABSTRACT Introduction: Vaccination with dendritic cells (DCs) has generally not fulfilled its promise in cancer immunotherapy due to ineffective translation of immune responses into clinical responses. A proposed reason for this is intrinsic immune regulatory mechanisms, such as regulatory T cells (Tregs). A metronomic regimen of cyclophosphamide (mCy) has been shown to selectively deplete Tregs. To test this in a clinical setting, we conducted a phase I trial to evaluate the feasibility and safety of vaccination with DCs transfected with mRNA in combination with mCy in patients with metastatic malignant melanoma (MM). In addition, clinical and immunological effect of the treatment was evaluated. Experimental design: Twenty-two patients were enrolled and treated with six cycles of cyclophosphamide 50 mg orally bi-daily for a week every second week (day 1–7). During the six cycles patients received at least 5 × 106 autologous DCs administered by intradermal (i.d.) injection in the week without chemotherapy. Patients were evaluated 12 and 27 weeks and every 3rd mo thereafter with CT scans according to RECIST 1.0. Blood samples for immune monitoring were collected at baseline, at the time of 4th and 6th vaccines. Immune monitoring consisted of IFNγ ELISpot assay, proliferation assay, and flow cytometry for enumeration of immune cell subsets. Results: Toxicity was manageable. Eighteen patients were evaluable after six cycles. Of these, nine patients had progressive disease as best response and nine patients achieved stable disease. In three patients minor tumor regression was observed. By IFNγ ELISpot and proliferation assay immune responses were seen in 6/17 and 4/17 patients, respectively; however, no correlation with clinical response was found. The percentage of Tregs was unchanged during treatment. Conclusion: Treatment with autologous DCs transfected with mRNA in combination with mCy was feasible and safe. Importantly, mCy did not alter the percentage of Tregs in our patient cohort. There was an indication of clinical benefit; however, more knowledge is needed in order for DCs to be exploited as a therapeutic option.

Dendritic cell vaccination in combination with docetaxel for patients with metastatic castration-resistant prostate cancer: A randomized phase II study

BACKGROUND AIMS We investigated whether the addition of an autologous dendritic cell-based cancer vaccine (DCvac) induces an immune response in patients with metastatic castration-resistant prostate cancer treated with docetaxel. METHODS Forty-three patients were randomized 1:1 to receive up to 10 cycles of docetaxel alone, 75 mg/m2 every 3 weeks or in combination with DCvac. Monocytes were harvested following a leukapheresis procedure, matured ex vivo and subsequently transfected with messenger RNA encoding multiple tumor-associated antigens (TAAs). DCvac was administered intradermally twice through treatment cycles 1-4 and once through treatment cycles 5-10. Immune cell composition and antigen-specific responses were analyzed using flow cytometry, ELISpot and delayed type hypersensitivity (DTH) tests. Toxicity was graded according to Common Terminology Criteria for Adverse Events version 3.0. Progression-free survival (PFS) and disease-specific survival (DSS) was calculated using the Kaplan-Meier method. RESULTS Prostate-specific antigen responses were similar in patients treated with docetaxel alone and combination therapy (58% versus 38%; P = 0.21). PFS and DSS were comparable: 5.5 versus 5.7 months (P = 0.62, log rank) and 21.9 versus 25.1 months (P = 0.60, log rank). Nine (50%) and 14 (78%) patients treated with docetaxel and DCvac had a TAA-specific or vaccine-specific immune response in the ELISpot and DTH analysis, respectively. Vaccine induced toxicity was limited to local reactions. Decline in myeloid-derived suppressor cells at the third treatment cycle was found to be an independent predictor of DSS. CONCLUSIONS The addition of DCvac was safe. Immune responses were detected in approximately half of the patients investigated.

Adoptive cell therapy with tumor infiltrating lymphocytes and intermediate dose IL-2 for metastatic melanoma

Meeting abstracts Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TILs) achieved impressive clinical results in several single institution Phase I/II clinical trials performed outside of Europe, and holds the promise to enter the mainstream of standard melanoma care in the near