Lotte Engell-Noerregaard

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.

CD8 T-cell Responses against Cyclin B1 in Breast Cancer Patients with Tumors Overexpressing p53

Purpose: This study aimed to examine CD8 T-cell reactivity in breast cancer patients against cyclin B1–derived peptides restricted by the human leukocyte antigen (HLA)-A2 molecule. Experimental Design: Peripheral blood mononuclear cells from 36 breast cancer patients were analyzed by enzyme-linked immunosorbent spot (ELISPOT) for the presence of T cells recognizing the cyclin B1–derived peptides CB9 (AKYLMELTM) and CB-P4 (AKYLMELCC), in addition to modified versions of CB9, CB9L2 (ALYLMELTM) and CB9M2 (AMYLMELTM), both of which display higher affinity to HLA-A2. Results: Twelve patients harbored a memory CD8 T-cell response against at least one of the peptides; strongest reactivity was detected against the CB9L2 peptide. Because the level of cyclin B1 has been shown to be influenced by the level of p53, which in turn is elevated in cancer cells because of point mutation, we analyzed the level of p53 protein in biopsies from the patients by immune histochemistry. Combined data showed that anti–cyclin B1 reactivity was predominantly detected in patients with tumors characterized by elevated expression of p53. Interestingly, no reactivity was detected against six peptides derived from the p53 protein. Conclusions: Our data support the notion of cyclin B1 as a prominent target for immunologic recognition in cancer patients harboring p53-mutated cancer cells. Because mutation of p53 is one of the most frequent genetic alterations in human cancers, this suggests that immunotherapy based on targeting of cyclin B1 is broadly applicable in a large proportion of cancer patients.

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.

FDG PET scans as evaluation of clinical response to dendritic cell vaccination in patients with malignant melanoma

BackgroundMeasurements of tumour metabolism by [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) have been successfully applied to monitor tumour response after chemo- and chemo-radiotherapy and may not have the same limitations as other morphological imaging techniques. In this study it is investigated whether FDG-PET might add information on the efficacy of immune therapy.Materials and methodsIn a retrospective analysis data from patients with advanced progressive melanoma, treated with DC vaccinations and evaluated by PET/CT scans at baseline as well as after 6 vaccinations were analysed. If a patient achieved stable disease according to RECIST, additional vaccinations were given. The PET scans were evaluated according to EORTC guidelines.ResultsPET/CT scans from 13 patients were evaluated. According to RECIST 3 patients achieved stable disease and 10 patients progressed. Interestingly, when evaluated by PET scans 2 patients had partial metabolic response and 1 patient had complete metabolic response of the 2 index lesions even though a new lesion appeared simultaneously. Ten patients were seen to have stable or progressive metabolic disease.ConclusionBy adding PET scans to the CT evaluation of patients treated with DC vaccines, a more detailed picture of the single lesions was found. This seems to improve the clinical evaluation of the treatment. The lack of correlation between the PET and CT scans suggests that some of the increases in target lesions seen in CT scans might be due to oedema or immune-infiltrates and not progression of the disease. Thus, further investigation into the contribution of PET scans to the evaluation of cancer immunotherapy is needed.

Influence of Metronomic Cyclophosphamide and Interleukine-2 alone or Combined on Blood Regulatory T Cells in Patients with Advanced Malignant Melanoma Treated with Dendritic Cell Vaccines

Background: Interleukine-2 and Cyclophosphamide are known to influence the circulating T cells and are used for immune manipulation in cancer patients. We analyzed the influence on the Treg population in three different dendritic cell (DC) vaccine trials including either concurrent interleukine-2 (IL-2), combined IL-2 and metronomic Cyclophosphamide (MCy), or only MCy.Methods: Melanoma patients received treatment with autologous DCs. IL-2 was applied in trial I and II and MCy was applied in trial I and III. Flowcytometry analysis was performed on fresh drawn blood-samples measuring CD4, CD25 and CD127.Results: In the trials where IL-2 was applied a marked increase in the proportion of CD4+CD25+CD127- Tregs from baseline to the 4th vaccine was observed. This was followed by a decrease, although not to baseline values. Additional analysis showed that the inhibitory function was predominant in the CD49d- subpopulation of Tregs which only increased slightly during treatment. The absolute lymphocyte count (ALC) and the number of CD4+ T cells also increased in these trials. In the trial where only MC was applied the Tregs remained stable throughout the trial whereas \\ decrease in the ALC and CD4+ T cells were observed.Conclusion: We found that adjuvant treatment with low doses of IL-2 during DC vaccination therapy causes a significant increase in blood level of classically defined, CD4+CD25+CD127-, Tregs but far less in the CD49d- subpopulation and that the use of MC was unable to reduce Treg blood level and unable to counteract IL-2 dependent increase in the CD4+CD25+CD127- T cells.These findings have implications for the clinical use of IL-2 as well as MCy as immune modulators.

Durable Clinical Responses and Long-Term Follow-Up of Stage III–IV Non-Small-Cell Lung Cancer (NSCLC) Patients Treated With IDO Peptide Vaccine in a Phase I Study—A Brief Research Report

Background: Long-term follow-up on a clinical trial of 15 stage III-IV NSCLC patients treated with an Indoleamine 2,3-Dioxygenase (IDO) peptide vaccine (NCT01219348). Methods: Fifteen HLA-A2-positive patients with stable stage III-IV NSCLC after standard chemotherapy were treated with subcutaneous vaccinations (100 μg IDO5 peptide, sequence ALLEIASCL, formulated in 900 μl Montanide) biweekly for 2.5 months and thereafter monthly until progression or up to 5 years. Here we report long-term clinical follow-up, toxicity and immunity. Results: Three of 15 patients are still alive corresponding to a 6-year overall survival of 20 %. Two patients continued monthly vaccinations for 5 years (56 vaccines). One of the two patients developed a partial response (PR) of target lesions in the liver 15 months after the first vaccine and has remained in PR ever since. The other patient had a solitary distant metastasis in a lymph node in retroperitoneum at baseline which normalized during treatment. All following evaluation scans during the treatment have been tumor free. The vaccine was well tolerated for all 5 years with no long-term toxicities registered. The third long-term surviving patient discontinued vaccinations after 11 months due to disease progression. Flow cytometry analyses of PBMCs from the two long-term responders demonstrated stable CD8+ and CD4+ T-cell populations during treatment. In addition, presence of IDO-specific T-cells was detected by IFN-γ Elispot in both patients at several time points during treatment. Conclusion: IDO peptide vaccination was well tolerated for administration up to 5years. Two of 15 patients are long-term responders with ongoing clinical response 6 years after 1st vaccination.

Long-lasting disease stabilization in the absence of toxicity in metastatic lung cancer patients vaccinated with an HLA-A2-restricted epitope derived from indoleamine 2,3 dioxygenase

Purpose: To investigate targeting of indoleamine 2,3 dioxygenase (IDO) enzyme using a synthetic peptide vaccine administered to patients with metastatic non–small cell lung cancer (NSCLC). Experimental Design: In a clinical phase I study, we treated 15 HLA-A2–positive patients with stage III– IV NSCLC in disease stabilization after standard chemotherapy. Patients were treated with imiquimod ointment and subcutaneous vaccinations (100 mg IDO5 peptide, sequence ALLEIASCL, formulated in 900 mL Montanide). Primary endpoint was toxicity. Clinical benefit and immunity were assessed as secondary endpoints. Results: No severe toxicity was observed. One patient developed a partial response (PR) after one year of vaccine treatment, whereas long-lasting stable disease (SD) � 8.5 months was demonstrated in another six patients. The median overall survival (OS) was 25.9 months. Patients demonstrated significant improved OS (P ¼ 0.03) when compared with the group of patients excluded because of HLA-A2 negativity. IDOspecific CD8 þ T-cell immunity was demonstrated by IFN-g Elispot and Tetramer staining. Fluorescenceactivated cell sorting analyses demonstrated a significant reduction of the Treg population (P ¼ 0.03) after the sixth vaccine (2.5 months) compared with pretreatment levels. Furthermore, expression of IDO was detected in nine of ten tumor biopsies by immunohistochemistry. High-performance liquid chromatography analyses of kynurenine/tryptophan (Kyn/Trp) ratio in sera were performed. In long-term analyses of two clinical responding patients, the ratio of Kyn/Trp remained stable. Conclusions: The vaccine was well tolerated with no severe toxicity occurring. A median OS of 25.9 months was demonstrated and long-lasting PRþSD was seen in 47% of the patients. Clin Cancer Res; 20(1); 221–32. � 2013 AACR.

Combined treatment with dendritic cell vaccine and low-dose cyclophosphamide in patients with malignant melanoma.

e13032 Background: Cancer vaccines based on dendritic cells (DC) have been tested in several clinical trials; in general, efficacy has been low, which might be due to inefficient immune activation....