Ulf Petrausch

Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells

IntroductionMalignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy.MethodsTo evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice.ResultsFAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice.ConclusionFAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells.

Hodgkin’s lymphoma in remission after first-line therapy: which patients need FDG–PET/CT for follow-up?

BACKGROUND The purpose of the study was to evaluate the impact of 2-[fluorine-18]fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET)/computed tomography (CT) during follow-up of patients with Hodgkins lymphoma. PATIENTS AND METHODS Patients in complete remission or an unconfirmed complete remission after first-line therapy who received FDG-PET/CT during their follow-up were analyzed retrospectively. Confirmatory biopsy was mandatory in case of recurrence. RESULTS Overall, 134 patients were analyzed. Forty-two (31.3%) patients had a recurrence. The positive predictive value of FDG-PET/CT was 0.98. Single-factor analysis identified morphological residual mass [P = 0.0005, hazard ratio (HR) 3.4, 95% confidence interval (CI) 1.7-6.6] and symptoms (P < 0.0001, HR 4.9, 95% CI 2.4-9.9) as significant risk factors for relapse. By multivariate analysis, morphological residual mass was the only significant risk factor for early follow-up (<24 months) (P = 0.0019, HR 7.6, 95% CI 2.1-27.3). Advanced stage (P = 0.0426, HR 3.6, 95% CI 1.1-12.3) and the presence of symptoms (P = 0.0009, HR = 14.6, 95% CI 3.0-69.7) were found to be significant risk factors for later follow-up (>24 months). CONCLUSIONS Asymptomatic patients without morphological residues and an early stage of disease do not need a routine FDG-PET/CT for follow-up. Asymptomatic patients with morphological residues should receive routine follow-up FDG-PET/CT for the first 24 months. Only patients with advanced initial stage do need a routine follow-up FDG-PET/CT beyond 24 months.

Effector memory and central memory NY-ESO-1-specific re-directed T cells for treatment of multiple myeloma

The cancer-testis antigen NY-ESO-1 is a potential target antigen for immune therapy expressed in a subset of patients with multiple myeloma. We generated chimeric antigen receptors (CARs) recognizing the immunodominant NY-ESO-1 peptide 157–165 in the context of HLA-A*02:01 to re-direct autologous CD8+ T cells towards NY-ESO-1+ myeloma cells. These re-directed T cells specifically lysed NY-ESO-1157–165/HLA-A*02:01-positive cells and secreted IFNγ. A total of 40% of CCR7− re-directed T cells had an effector memory phenotype and 5% a central memory phenotype. Based on CCR7 cell sorting, effector and memory CAR-positive T cells were separated and CCR7+ memory cells demonstrated after antigen-specific re-stimulation downregulation of CCR7 as sign of differentiation towards effector cells accompanied by an increased secretion of memory signature cytokines such as IL-2. To evaluate NY-ESO-1 as potential target antigen, we screened 78 bone marrow biopsies of multiple myeloma patients where NY-ESO-1 protein was found to be expressed by immunohistochemistry in 9.7% of samples. Adoptively transferred NY-ESO-1-specific re-directed T cells protected mice against challenge with endogenously NY-ESO-1-positive myeloma cells in a xenograft model. In conclusion, re-directed effector- and central memory T cells specifically recognized NY-ESO-1157–165/ HLA-A*02:01-positive cells resulting in antigen-specific functionality in vitro and in vivo.

Risk-adapted FDG–PET/CT-based follow-up in patients with diffuse large B-cell lymphoma after first-line therapy

BACKGROUND The purpose of this study was to evaluate the impact of 2-[fluorine-18]fluoro-2-deoxy-D-glucose-positron emission tomography/computed tomography (FDG-PET/CT) during follow-up of patients with diffuse large B-cell lymphoma (DLBCL) being in complete remission or unconfirmed complete remission after first-line therapy. PATIENTS AND METHODS DLBCL patients receiving FDG-PET/CT during follow-up were analyzed retrospectively. Confirmatory biopsy was mandatory in cases of suspected disease recurrence. RESULTS Seventy-five patients were analyzed and 23 (30%) had disease recurrence. The positive predictive value (PPV) of FDG-PET/CT was 0.85. Patients >60 years [P = 0.036, hazard ratio (HR) = 3.82, 95% confidence interval (CI) 1.02-7.77] and patients with symptoms indicative of a relapse (P = 0.015; HR = 4.1; 95% CI 1.20-14.03) had a significantly higher risk for relapse. A risk score on the basis of signs of relapse, age >60 years, or a combination of these factors identified patients at high risk for recurrence (P = 0.041). CONCLUSIONS FDG-PET/CT detects recurrent DLBCL after first-line therapy with high PPV. However, it should not be used routinely and if only in selected high-risk patients to reduce radiation burden and costs. On the basis of our retrospective data, FDG-PET/CT during follow-up is indicated for patients <60 years with clinical signs of relapse and in patients >60 years with and without clinical signs of relapse.

Disruption of TGF-β Signaling Prevents the Generation of Tumor-Sensitized Regulatory T Cells and Facilitates Therapeutic Antitumor Immunity

Regulatory T (Treg) cells represent a major roadblock to the induction of antitumor immunity through vaccine approaches. TGF-β is a cytokine implicated in the generation and maintenance of Treg cells, as well as in their suppressive function. These experiments examined whether the generation of tumor-sensitized Treg cells was TGF-β dependent and evaluated whether TGF-β produced by Treg cells blocked the priming of tumor-specific T cells in vaccinated reconstituted lymphopenic mice. We show that tumor-sensitized Treg cells (CD25+/FoxP3+) obtained from tumor-bearing mice block the generation of tumor-specific T cells in reconstituted lymphopenic mice. Strikingly, this suppression is absent if tumor-sensitized Treg cells are acquired from tumor-bearing mice expressing the dominant-negative TGFβRII in T cells. This loss of suppression was a result of the crucial role of TGF-β in generating tumor-sensitized Treg cells, and not due to the insensitivity of naive or tumor-primed effector T cells to the direct suppressive influence of TGF-β. We conclude that blocking TGF-β in a tumor-bearing host can inhibit the induction of highly suppressive tumor-sensitized Treg cells. These data suggest that an integrative strategy combining “up-front” Treg cell ablation followed by vaccination and TGF-β blockade may limit generation of new tumor-sensitized Treg cells and improve the generation of therapeutic immune responses in patients with cancer.

Re-directed T cells for the treatment of fibroblast activation protein (FAP)-positive malignant pleural mesothelioma (FAPME-1)

BackgroundAsbestos is the main cause of MPM in industrialized countries. Even since asbestos is banned in most developed countries, the peak wave of MPM incidence is anticipated for the next years due to the long latency of asbestos induced MPM. MPM patients not eligible for surgical procedures like decortication or pleuro-pneumectomie have a median survival of 12 months with palliative chemotherapy. Therefore, new therapeutic approaches are of crucial need in this clinical situation.Methods/designThis is a phase I trial for patients with malignant pleural mesothelioma with pleural effusion testing the safety of a fixed single dose of 1x106 adoptively transferred FAP-specific re-directed T cells given directly in the pleural effusion. Lymphocytes will be taken 21 days before transfer from peripheral blood. CD8 positive T cells will be isolated and re-programmed by retroviral transfer of a chimeric antigen receptor recognizing FAP which serves as target structure in MPM. At day 0 of the protocol, re-directed T cells will be injected in the pleural effusion and patients will be monitored for 48h under intermediate care conditions. AE, SAE, SADR and SUSAR will be monitored for 35 days and evaluated by an independent safety board to define any dose limiting toxicity (DLT). No further patient can be treated before the previous patient passed day 14 after T cell transfer. The protocol will be judged as save when no DLT occurred in the first 3 patients, or 1 DLT in 6 patients. Secondary objectives are feasibility and immune monitoring.DiscussionAdoptive T cell transfer is a new and rapidly expanding branch of immunotherapies focusing on cancer treatment. Recently, objective responses could be observed in patients with chronic lymphatic leukemia treated with adoptively transferred CD19-specific re-directed T cells. The choice of the target antigen determines the possible on-target off-tissue toxicity of such approaches. There are reports of severe toxicity in patients who received T cells intravenously due to unexpected expression of the target antigen (on-target) in other tissues than the tumor (off-tissue). To minimize the risk of on-target off-tissue toxicity and to maximize the on-target anti-tumor effect we propose a clinical protocol with loco-regional administration of re-directed T cells. FAP-specific T cells will be directly injected in the pleural effusion of patients with MPM.Trial registrationClinicalTrials.gov (NCT01722149)

Phenotype and Functional Characterization of Long-term gp100-Specific Memory CD8+ T Cells in Disease-Free Melanoma Patients Before and After Boosting Immunization

Purpose: Effective cancer vaccines must both drive a strong CTL response and sustain long-term memory T cells capable of rapid recall responses to tumor antigens. We sought to characterize the phenotype and function of gp100 peptide-specific memory CD8+ T cells in melanoma patients after primary gp100209-2M immunization and assess the anamnestic response to boosting immunization. Experimental Design: Eight-color flow cytometry analysis of gp100-specific CD8+ T cells was done on peripheral blood mononuclear cells collected shortly after the primary vaccine regimen, 12 to 24 months after primary vaccination, and after boosting immunization. The anamnestic response was assessed by comparing the frequency of circulating gp100-specific T cells before and after boosting. Gp100 peptide-induced in vitro functional avidity and proliferation responses and melanoma-stimulated T-cell CD107 mobilization were compared for cells from all three time points for multiple patients. Results: The frequency of circulating gp100-specific memory CD8+ T cells was comparable with cytomegalovirus-specific and FLU-specific T cells in the same patients, and the cells exhibited anamnestic proliferation after boosting. Their phenotypes were not unique, and individual patients exhibited one of two distinct phenotype signatures that were homologous to either cytomegalovirus-specific or FLU-specific memory T cells. Gp100-specific memory T cells showed some properties of competent memory T cells, such as heightened in vitro peptide-stimulated proliferation and increase in central memory (TCM) differentiation when compared with T-cell responses measured after the primary vaccine regimen. However, they did not acquire enhanced functional avidity usually associated with competent memory T-cell maturation. Conclusions: Although vaccination with class I–restricted melanoma peptides alone can break tolerance to self-tumor antigens, it did not induce fully competent memory CD8+ T cells—even in disease-free patients. Data presented suggest other vaccine strategies will be required to induce functionally robust long-term memory T cells.

Increased Frequency of Suppressive Regulatory T Cells and T Cell-Mediated Antigen Loss Results in Murine Melanoma Recurrence

Therapeutic treatment of large established tumors using immunotherapy has yielded few promising results. We investigated whether adoptive transfer of tumor-specific CD8+ T cells, together with tumor-specific CD4+ T cells, would mediate regression of large established B16BL6-D5 melanomas in lymphopenic Rag1−/− recipients devoid of regulatory T cells. The combined adoptive transfer of subtherapeutic doses of both TRP1-specific TCR transgenic Rag1−/− CD4+ T cells and gp100-specific TCR transgenic Rag1−/− CD8+ T cells into lymphopenic recipients, who received vaccination, led to regression of large (100–400 mm2) melanomas. The same treatment strategy was ineffective in lymphoreplete wild-type mice. Twenty-five percent of mice (15/59) had tumors recur (15–180 d postregression). Recurrent tumors were depigmented and had decreased expression of gp100, the epitope targeted by the CD8+ T cells. Mice with recurrent melanoma had increased CD4+Foxp3+ TRP1-specific T cells compared with mice that did not show evidence of disease. Importantly, splenocytes from mice with recurrent tumor were able to suppress the in vivo therapeutic efficacy of splenocytes from tumor-free mice. These data demonstrate that large established tumors can be treated by a combination of tumor-specific CD8+ and CD4+ T cells. Additionally, recurrent tumors exhibited decreased Ag expression, which was accompanied by conversion of the therapeutic tumor-specific CD4+ T cell population to a Foxp3+CD4+ regulatory T cell population.

Cancer Immunotherapy: The Role Regulatory T Cells Play and What Can be Done to Overcome their Inhibitory Effects

Since multiple lines of experimental and clinical data clearly identified regulatory T cells as an integral part of the immune response, these cells have become a major focus of investigation in tumor immunology. Regulatory T cells are in place to dampen ongoing immune responses and to prevent autoimmunity, but they also have profound effects in blocking therapeutic anti-tumor activity. Therefore regulatory T cells are seen as a major hurdle that must be overcome in order for cancer immunotherapy to reach its therapeutic potential. Regulatory T cells are heterogeneous with sub-populations that exhibit distinct functional features. Here we will review the individual sub-populations in regards to their mode of action and their potential impact on blocking anti-tumor immunity. Approaches to measure function and frequency of regulatory T cells in model systems and clinical trails will be discussed. Finally, we will describe possible ways to interfere with regulatory T cell-mediated immune suppression with the focus on recent pre-clinical and clinical findings.

Daily Pomegranate Intake Has No Impact on PSA Levels in Patients with Advanced Prostate Cancer - Results of a Phase IIb Randomized Controlled Trial.

Pomegranate has been shown to prolong PSA doubling time in early prostate cancer, but no data from a placebo controlled trial has been published yet. The objective of this study was to prospectively evaluate the impact of pomegranate juice in patients with prostate cancer. We conducted a phase IIb, double blinded, randomized placebo controlled trial in patients with histologically confirmed prostate cancer. Only patients with a PSA value ≥ 5ng/ml were included. The subjects consumed 500 ml of pomegranate juice or 500 ml of placebo beverage every day for a 4 week period. Thereafter, all patients received 250 ml of the pomegranate juice daily for another 4 weeks. PSA values were taken at baseline, day 14, 28 and on day 56. The primary endpoint was the detection of a significant difference in PSA serum levels between the groups after one month of treatment. Pain scores and adherence to intervention were recorded using patient diaries. 102 patients were enrolled. The majority of patients had castration resistant prostate cancer (68%). 98 received either pomegranate juice or placebo between October 2008 and May 2011. Adherence to protocol was good, with 94 patients (96%) completing the first period and 87 patients (89%) completing both periods. No grade 3 or higher toxicities occurred within the study. No differences were detected between the two groups with regard to PSA kinetics and pain scores. Consumption of pomegranate juice as an adjunct intervention in men with advanced prostate cancer does not result in significant PSA declines compared to placebo.