We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1-restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1-restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer-based broad-spectrum immunotherapy of malignant disease.

BackgroundAlthough commonly utilized interventions, no studies have directly compared the effectiveness of cervical and thoracic manipulation to mobilization and exercise in individuals with cervicogenic headache (CH). The purpose of this study was to compare the effects of manipulation to mobilization and exercise in individuals with CH.MethodsOne hundred and ten participants (n = 110) with CH were randomized to receive both cervical and thoracic manipulation (n = 58) or mobilization and exercise (n = 52). The primary outcome was headache intensity as measured by the Numeric Pain Rating Scale (NPRS). Secondary outcomes included headache frequency, headache duration, disability as measured by the Neck Disability Index (NDI), medication intake, and the Global Rating of Change (GRC). The treatment period was 4 weeks with follow-up assessment at 1 week, 4 weeks, and 3 months after initial treatment session. The primary aim was examined with a 2-way mixed-model analysis of variance (ANOVA), with treatment group (manipulation versus mobilization and exercise) as the between subjects variable and time (baseline, 1 week, 4 weeks and 3 months) as the within subjects variable.ResultsThe 2X4 ANOVA demonstrated that individuals with CH who received both cervical and thoracic manipulation experienced significantly greater reductions in headache intensity (p < 0.001) and disability (p < 0.001) than those who received mobilization and exercise at a 3-month follow-up. Individuals in the upper cervical and upper thoracic manipulation group also experienced less frequent headaches and shorter duration of headaches at each follow-up period (p < 0.001 for all). Additionally, patient perceived improvement was significantly greater at 1 and 4-week follow-up periods in favor of the manipulation group (p < 0.001).ConclusionsSix to eight sessions of upper cervical and upper thoracic manipulation were shown to be more effective than mobilization and exercise in patients with CH, and the effects were maintained at 3 months.Trial registrationNCT01580280 April 16, 2012.

Summary:The purpose of this study was to evaluate feasibility and efficacy of Rituximab included into a sequential salvage protocol for CD20 + B-NHL in relapse or induction failure. Twenty-seven patients with CD20 + B-NHL in relapse or induction failure received Rituximab combined with DexaBEAM (R-DexaBEAM) for stem cell mobilization. Additional ex vivo selection of CD34-positive cells was performed using the CliniMacs device. Two doses of Rituximab were included in the high-dose therapy regimen (HDT). R-DexaBEAM was well toler-

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Malignant transformation of normal cells is frequently correlated with the involvement of so called tumor-associated antigens (TAA). Such proteins, that are often overexpressed in tumor cells, can be recognized by cytotoxic CD8+ T cells (CTL) if presented as peptides on MHC (Major-Histocompatibility-Complex)-class I molecules. Due to self-tolerance mechanisms, the peripheral T cell repertoire is devoid of efficient TAA-specific, tumor-reactive CTL with high affinity, limiting the successful development of antigen-specific immunotherapeutic strategies based on such tumor-reactive T cells. The aim of this project is the preclinical development of an adoptive immunotherapy against p53-positive tumors. The p53 tumor-suppressor protein is an antigen, which is markedly upregulated in multiple tumors. Such overexpression of p53 correlates with the presence of mutated variants of p53, which inactivate p53’s normal function as tumor-suppressor. Tumors with wildtype (wt) p53 also exhibit abnormalities in p53 expression, metabolism, and function, which arise from alterations of an impressive repertoire of p53-interacting gene products. Hence, perturbation of p53 regulatory pathways and metabolism occurs in most, if not all, types of human malignancies and makes class I MHC bound peptides naturally processed from the wt p53 protein almost universal targets for tumor-specific CD8+ CTL. The generation of CTL, bearing a high-affinity p53-specific T-cell receptor (TCR) reactive against such peptide/MHC-class I complexes presented on tumors, would display a promising strategy for cancer immunotherapy. Through adoptive transfer of autologous PBMCs, retrovirally transduced with genes encoding such TCR, self-tolerance against p53 in tumor patients could be circumvented leading to selective destruction of p53-expressing tumors without harming healthy tissue. Previous studies have described strategies to circumvent self-tolerance against p53 using HLA-A2.1 transgenic mice. Immunization of such mice with peptides derived from human p53 has generated efficient high-affinity p53A2.1-specific CTL clones. Moreover the TCR from one of these clones that specifically recognizes a wt p53 peptide (aa 264–272) could be cloned for the transduction into human T-cells. We could show that the introduction of this p53(264–272)A2.1-specific-TCR into human PBMCs via retroviral transduction produced effective and high affine T cells of high affinity capable of recognizing and lysing p53+ tumor cells while sparing normal p53-expressing cells. Apart from CD8+ T cells, p53(264–272)A2.1-specific-TCR-transduced CD4+ helper T cells (Th) too were able to recognize peptide-loaded p53(264–272)A2-complexes as well as p53/HLA-A2-positive tumor cells. Moreover both T cell subsets were cooperative and interacted synergistically with dendritic cell intermediates and tumor targets. The adoptive transfer of such gene-modified T cells could display a highly effective therapy against p53-positive tumors. To demonstrate the therapeutic potential of p53(264–272)/HLA-A2-specific TCR transgenic cells for a possible clinical translation, we have initiated preclinical studies for the adoptive transfer of such cells. Here, we demonstrate the function and safety in vitro and in vivo of a retroviral GMP-suitable vector construct coding for the p53(264–272)/HLA-A2-specific TCR designed for clinical application.