Publications by authors named "Thomas Blankenstein"

111 Publications

Long-term in vitro expansion ensures increased yield of central memory T cells as perspective for manufacturing challenges.

Int J Cancer 2021 Feb 18. Epub 2021 Feb 18.

Experimental and Clinical Research Center, Berlin, Germany.

Adoptive T cell therapy (ATT) has revolutionized the treatment of cancer patients. A sufficient number of functional T cells are indispensable for ATT efficacy; however, several ATT dropouts have been reported due to T cell expansion failure or lack of T cell persistence in vivo. With the aim of providing ATT also to those patients experiencing insufficient T cell manufacturing via standard protocol, we evaluated if minimally manipulative prolongation of in vitro expansion (long-term [LT] >3 weeks with IL-7 and IL-15 cytokines) could result in enhanced T cell yield with preserved T cell functionality. The extended expansion resulted in a 39-fold increase of murine CD8 T central memory cells (Tcm). LT expanded CD8 and CD4 Tcm cells retained a gene expression profile related to Tcm and T memory stem cells (Tscm). In vivo transfer of LT expanded Tcm revealed persistence and antitumor capacity. We confirmed our in vitro findings on human T cells, on healthy donors and diffuse large B cell lymphoma patients, undergoing salvage therapy. Our study demonstrates the feasibility of an extended T cell expansion as a practicable alternative for patients with insufficient numbers of T cells after the standard manufacturing process thereby increasing ATT accessibility.
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http://dx.doi.org/10.1002/ijc.33523DOI Listing
February 2021

Thyrotropin Receptor-Specific Lymphocytes in Adenovirus-TSHR-Immunized Native and Human Leukocyte Antigen-DR3-Transgenic Mice and in Graves' Disease Patient Blood.

Thyroid 2021 Feb 8. Epub 2021 Feb 8.

Procorde-Advancecor, Martinsried, Germany.

Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyrotropin receptor (TSHR)-specific lymphocytes in mouse models of Graves' disease, including Graves' patient-specific immunotype human leukocyte antigen (HLA)-DR3, and in frozen and thawed Graves' patient blood samples. Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154-expressing cells were enriched, expanded , and analyzed for binding of peptide-major histocompatibility complex (MHC) II multimers ("tetramers," immunotype H2-IA). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon-γ, and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in reliable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25, CD127). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves' patients' thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha from thawed PBMCs occurred at the expected levels. Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice and in thawed human PBMCs from patients with Graves' disease. These assays may contribute to measure both disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves' disease.
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http://dx.doi.org/10.1089/thy.2020.0338DOI Listing
February 2021

Regulation of LCoR and RIP140 expression in cervical intraepithelial neoplasia and correlation with CIN progression and dedifferentiation.

J Cancer Res Clin Oncol 2020 Jul 10;146(7):1847-1855. Epub 2020 Mar 10.

Department of Obstetrics and Gynecology, University Hospital, LMU Munich, 80337, Munich, Germany.

Purpose: Ligand-dependent corepressor (LCoR) and receptor-interacting protein 140 (RIP140/NRIP1) play an important role in the regulation of multiple oncogenic signaling pathways and the development of cancer. LCoR and RIP140 form a nuclear complex in breast cancer cells and are of prognostic value in further prostate and cervical cancer. The purpose of this study was to analyze the regulation of these proteins in the development of cervical intraepithelial neoplasia (CIN I-III).

Methods: Immunohistochemical analysis was obtained to quantify RIP140 and LCoR expression in formalin-fixed paraffin embedded tissue sections of cervical intraepithelial neoplasia samples. Tissue (n = 94) was collected from patients treated in the Department of Gynecology and Obstetrics, Ludwig-Maximilians-University of Munich, Germany, between 2002 and 2014. Correlations of expression levels with clinical outcome were carried out to assess for prognostic relevance in patients with CIN2 progression. Kruskal-Wallis test and Mann-Whitney U test were used for data analysis.

Results: Nuclear LCoR overexpression correlates significantly with CIN II progression. Nuclear RIP140 expression significantly increases and nuclear LCoR expression decreases with higher grading of cervical intraepithelial neoplasia. Cytoplasmic RIP140 expression is significantly higher in CIN III than in CIN I or CIN II.

Conclusion: A decrease of nuclear LCoR expression in line with an increase of dedifferentiation of CIN can be observed. Nuclear LCoR overexpression correlates with CIN II progression indicating a prognostic value of LCoR in cervical intraepithelial neoplasia. Nuclear and cytoplasmic RIP140 expression increases significantly with higher grading of cervical intraepithelial neoplasia underlining its potential role in the development of pre-cancerous lesions. These findings support the relevance of LCoR and RIP140 in the tumorigenesis indicating a possible role of LCoR and RIP140 as targets for novel therapeutic approaches in cervical intraepithelial neoplasia and cervical cancer.
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http://dx.doi.org/10.1007/s00432-020-03178-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256097PMC
July 2020

[Often not requiring therapy].

MMW Fortschr Med 2020 01;162(1):60-61

Klinikum der LMU München, Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Maistr. 11, D-80337, München, Deutschland.

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http://dx.doi.org/10.1007/s15006-020-0063-1DOI Listing
January 2020

Identification and ranking of recurrent neo-epitopes in cancer.

BMC Med Genomics 2019 11 27;12(1):171. Epub 2019 Nov 27.

Core Unit Bioinformatics, Berlin Institute of Health, Charitéplatz 1, Berlin, 10117, Germany.

Background: Immune escape is one of the hallmarks of cancer and several new treatment approaches attempt to modulate and restore the immune system's capability to target cancer cells. At the heart of the immune recognition process lies antigen presentation from somatic mutations. These neo-epitopes are emerging as attractive targets for cancer immunotherapy and new strategies for rapid identification of relevant candidates have become a priority.

Methods: We carefully screen TCGA data sets for recurrent somatic amino acid exchanges and apply MHC class I binding predictions.

Results: We propose a method for in silico selection and prioritization of candidates which have a high potential for neo-antigen generation and are likely to appear in multiple patients. While the percentage of patients carrying a specific neo-epitope and HLA-type combination is relatively small, the sheer number of new patients leads to surprisingly high reoccurence numbers. We identify 769 epitopes which are expected to occur in 77629 patients per year.

Conclusion: While our candidate list will definitely contain false positives, the results provide an objective order for wet-lab testing of reusable neo-epitopes. Thus recurrent neo-epitopes may be suitable to supplement existing personalized T cell treatment approaches with precision treatment options.
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http://dx.doi.org/10.1186/s12920-019-0611-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882202PMC
November 2019

SigsPack, a package for cancer mutational signatures.

BMC Bioinformatics 2019 Sep 2;20(1):450. Epub 2019 Sep 2.

Core Unit Bioinformatics, Berlin Institute of Health, Charitéplatz 1, Berlin, 10117, Germany.

Background: Mutational signatures are specific patterns of somatic mutations introduced into the genome by oncogenic processes. Several mutational signatures have been identified and quantified from multiple cancer studies, and some of them have been linked to known oncogenic processes. Identification of the processes contributing to mutations observed in a sample is potentially informative to understand the cancer etiology.

Results: We present here SigsPack, a Bioconductor package to estimate a sample's exposure to mutational processes described by a set of mutational signatures. The package also provides functions to estimate stability of these exposures, using bootstrapping. The performance of exposure and exposure stability estimations have been validated using synthetic and real data. Finally, the package provides tools to normalize the mutation frequencies with respect to the tri-nucleotide contents of the regions probed in the experiment. The importance of this effect is illustrated in an example.

Conclusion: SigsPack provides a complete set of tools for individual sample exposure estimation, and for mutation catalogue & mutational signatures normalization.
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http://dx.doi.org/10.1186/s12859-019-3043-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720940PMC
September 2019

Localization-associated immune phenotypes of clonally expanded tumor-infiltrating T cells and distribution of their target antigens in rectal cancer.

Oncoimmunology 2019;8(6):e1586409. Epub 2019 Mar 24.

Department of Hematology, Oncology, and Tumor Immunology, Charité - Universitätsmedizin Berlin (CVK), Berlin, Germany.

The degree and type of T cell infiltration influence rectal cancer prognosis regardless of classical tumor staging. We asked whether clonal expansion and tumor infiltration are restricted to selected-phenotype T cells; which clones are accessible in peripheral blood; and what the spatial distribution of their target antigens is. From five rectal cancer patients, we isolated paired tumor-infiltrating T cells (TILs) and T cells from unaffected rectum mucosa (T) using 13-parameter FACS single cell index sorting. TCRαβ sequences, cytokine, and transcription factor expression were determined with single cell sequencing. TILs and T occupied distinct phenotype compartments and clonal expansion predominantly occurred within CD8 T cells. Expanded TIL clones identified by paired TCRαβ sequencing and exclusively detectable in the tumor showed characteristic PD-1 and TIM-3 expression. TCRβ repertoire sequencing identified 49 out of 149 expanded TIL clones circulating in peripheral blood and 41 (84%) of these were PD-1 TIM-3. To determine whether clonal expansion of predominantly tumor-infiltrating T cell clones was driven by antigens uniquely presented in tumor tissue, selected TCRs were reconstructed and incubated with cells isolated from corresponding tumor or unaffected mucosa. The majority of clones exclusively detected in the tumor recognized antigen at both sites. In summary, rectal cancer is infiltrated with expanded distinct-phenotype T cell clones that either i) predominantly infiltrate the tumor, ii) predominantly infiltrate the unaffected mucosa, or iii) overlap between tumor, unaffected mucosa, and peripheral blood. However, the target antigens of predominantly tumor-infiltrating TIL clones do not appear to be restricted to tumor tissue.
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http://dx.doi.org/10.1080/2162402X.2019.1586409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492980PMC
March 2019

Interferon-γ Receptor Signaling in Dendritic Cells Restrains Spontaneous Proliferation of CD4 T Cells in Chronic Lymphopenic Mice.

Front Immunol 2019 7;10:140. Epub 2019 Feb 7.

Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany.

In lymphopenic mice, T cells become activated and undergo lymphopenia-induced proliferation (LIP). However, not all T cells are equally sensitive to lymphopenia. Several lymphopenia-insensitive T cell clones were described and their non-responsiveness was mainly attributed to clone-specific properties. Here, we provide evidence for an additional, host-dependent mechanism restraining LIP of lymphopenia-insensitive CD4 T cells. We show that such cells undergo LIP in lymphopenic mice lacking IFN-γ receptor (IFN-γR) expression, a process, which is promoted by the autocrine action of T cell-derived IFN-γ. Additionally, LIP of lymphopenia-insensitive CD4 T cells requires an intact microflora and is accompanied by the massive accumulation of IL-6 and dendritic cells (DCs). Consistent with these results, IL-6 neutralization and the DC-specific restoration of IFN-γR expression are both sufficient to restrict LIP. Hence, the insensitivity of CD4 T cells to lymphopenia relies on cell-intrinsic properties and a complex interplay between the commensal microflora, IL-6, IFN-γR DCs, and T cell-derived IFN-γ.
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http://dx.doi.org/10.3389/fimmu.2019.00140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374634PMC
January 2020

Effective NY-ESO-1-specific MHC II-restricted T cell receptors from antigen-negative hosts enhance tumor regression.

J Clin Invest 2019 01 10;129(1):324-335. Epub 2018 Dec 10.

Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells is a promising approach in cancer therapy but needs improvement for more effective treatment of solid tumors. While most clinical approaches have focused on CD8+ T cells, the importance of CD4+ T cells in mediating tumor regression has become apparent. Regarding shared (self) tumor antigens, it is unclear whether the human CD4+ T cell repertoire has been shaped by tolerance mechanisms and lacks highly functional TCRs suitable for therapy. Here, TCRs against the tumor-associated antigen NY-ESO-1 were isolated either from human CD4+ T cells or from mice that express a diverse human TCR repertoire with HLA-DRA/DRB1*0401 restriction and are NY-ESO-1 negative. NY-ESO-1-reactive TCRs from the mice showed superior recognition of tumor cells and higher functional activity compared with TCRs from humans. We identified a candidate TCR, TCR-3598_2, which was expressed in CD4+ T cells and caused tumor regression in combination with NY-ESO-1-redirected CD8+ T cells in a mouse model of adoptive T cell therapy. These data suggest that MHC II-restricted TCRs against NY-ESO-1 from a nontolerant nonhuman host are of optimal affinity and that the combined use of MHC I- and II-restricted TCRs against NY-ESO-1 can make adoptive T cell therapy more effective.
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http://dx.doi.org/10.1172/JCI120391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307948PMC
January 2019

Oncogene-specific T cells fail to eradicate lymphoma-initiating B cells in mice.

Blood 2018 08 12;132(9):924-934. Epub 2018 Jul 12.

Institute of Immunology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany.

To date, little is known about the interaction between (pre-)malignant B cells and T cells. We generated transgenic mice that allow B cell-specific induction of the oncogene SV40 large T-antigen (TAg) to analyze the role of oncogene-specific T cells during sporadic B-cell lymphoma development. Constitutive TAg expression in CD19-Cre × LoxP-Tag mice resulted in TAg-tolerant CD8 T cells and development of B-cell lymphomas. In contrast, CD19-CreER × LoxP-Tag mice retained TAg-competent CD8 T cells at time of oncogene induction and TAg expression in few B cells of adult mice resulted in exceptionally rare lymphoma formation late in life. Increased lymphoma incidence in the absence of TAg-specific T cells suggested T cell-mediated inhibition of lymphoma progression. However, TAg-initiated B cells were not eliminated by T cells and detected long term. Our results demonstrate a failure of the immune system to eradicate lymphoma-initiating B cells, retaining the risk of lymphoma development.
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http://dx.doi.org/10.1182/blood-2018-02-834036DOI Listing
August 2018

Targeting Merkel Cell Carcinoma by Engineered T Cells Specific to T-Antigens of Merkel Cell Polyomavirus.

Clin Cancer Res 2018 08 18;24(15):3644-3655. Epub 2018 Apr 18.

Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.

The causative agent of most cases of Merkel cell carcinoma (MCC) has been identified as the Merkel cell polyomavirus (MCV). MCV-encoded T antigens (Tag) are essential not only for virus-mediated tumorigenesis but also for maintaining MCC cell lines MCV Tags are thus an appealing target for viral oncoprotein-directed T-cell therapy for MCC. With this study, we aimed to isolate and characterize Tag-specific T-cell receptors (TCR) for potential use in gene therapy clinical trials. T-cell responses against MCV Tag epitopes were investigated by immunizing transgenic mice that express a diverse human TCR repertoire restricted to HLA-A2. Human lymphocytes genetically engineered to express Tag-specific TCRs were tested for specific reactivity against MCC cell lines. The therapeutic potential of Tag-specific TCR gene therapy was tested in a syngeneic cancer model. We identified naturally processed epitopes of MCV Tags and isolated Tag-specific TCRs. T cells expressing these TCRs were activated by HLA-A2-positive cells loaded with cognate peptide or cells that stably expressed MCV Tags. We showed cytotoxic potential of T cells engineered to express these TCRs and demonstrated regression of established tumors in a mouse model upon TCR gene therapy. Our findings demonstrate that MCC cells can be targeted by MCV Tag-specific TCRs. Although recent findings suggest that approximately half of MCC patients benefit from PD-1 pathway blockade, additional patients may benefit if their endogenous T-cell response can be augmented by infusion of transgenic MCV-specific T cells such as those described here. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082703PMC
August 2018

ERAP1-Dependent Antigen Cross-Presentation Determines Efficacy of Adoptive T-cell Therapy in Mice.

Cancer Res 2018 06 20;78(12):3243-3254. Epub 2018 Mar 20.

Institute of Biochemistry, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany.

Cytotoxic T lymphocytes can reject established tumors if their target peptide is efficiently presented by MHC class I molecules (pMHC-I) on the surface of cancerous cells. Therapeutic success upon adoptive T-cell transfer (ATT), however, requires additional cross-presentation of the same pMHC-I on noncancerous cells. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that customizes the N-terminus of proteasome-generated peptides so they can be loaded onto MHC-I molecules in the endoplasmic reticulum (ER). We show here that ERAP1 is critically involved in the process of tumor rejection and assumes a dual role by independently operating on both sides. Direct presentation of two MHC-I-restricted epitopes of a cancer-driving transplantation rejection antigen through ERAP1 moderately affected tumor rejection by adoptively transferred T-cell receptor gene-modified T cells in each case. ERAP1 expression by antigen cross-presenting cells of the ATT recipients was critical for expansion of therapeutic monospecific T cells and correlated with tumor rejection. Specifically, lack of ERAP1 expression in the ATT recipient's noncancerous cells enabled progression of pMHC-I-positive, IFNγ-responsive tumors, despite the presence of antigen-specific functional cytotoxic T lymphocytes. These data reveal a decisive role for ERAP1 in T-cell-mediated tumor rejection and will enhance the choice of MHC-I-restricted epitopes targeted by adoptive T-cell transfer. This study demonstrates a role of ERAP1 in the efficacy of adoptive T-cell transfer and has potential to improve personalized T-cell therapy for solid tumors. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1946DOI Listing
June 2018

[Changes to cervical cancer screening in Germany].

MMW Fortschr Med 2018 Feb;160(2):46-51

Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Klinikum der Universität München, Campus Großhadern, Marchioninistraße 15, D-81377, München, Deutschland.

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http://dx.doi.org/10.1007/s15006-018-0152-6DOI Listing
February 2018

A Transgenic Dual-Luciferase Reporter Mouse for Longitudinal and Functional Monitoring of T Cells .

Cancer Immunol Res 2018 01 19;6(1):110-120. Epub 2017 Dec 19.

Experimental and Clinical Research Center (ECRC), Berlin, Germany.

Adoptive T-cell therapy (ATT) efficacy is limited when targeting large solid tumors. The evaluation of ATT outcomes using accessory treatment would greatly benefit from an monitoring tool, allowing the detection of functional parameters of transferred T cells. Here, we generated transgenic bioluminescence imaging of T cells (BLITC) mice expressing an NFAT-dependent click-beetle luciferase and a constitutive luciferase, which supports concomitant analysis of migration and activation of T cells. Rapid transferability of our system to preestablished tumor models was demonstrated in the SV40-large T antigen model via both crossbreeding of BLITC mice into a T-cell receptor (TCR)-transgenic background and TCR transduction of BLITC T cells. We observed rapid tumor infiltration of BLITC CD8 T cells followed by a burst-like activation that mirrored rejection kinetics. Using the BLITC reporter in the clinically relevant H-Y model, we performed female to male transfers and detected H-Y-specific alloreactivity (graft-versus-host disease) In an H-Y solid tumor model, we found migration of adoptively transferred H-Y TCR-transgenic CD4 T cells into the tumor, marked by transient activation. This suggests a rapid inactivation of infiltrating T cells by the tumor microenvironment, as confirmed by their expression of inhibitory receptors. In summary, the BLITC reporter system facilitates analysis of therapeutic parameters for ATT, is rapidly transferable to models of interest not restricted to tumor research, and is suitable for rapid screening of TCR clones for tumor rejection kinetics, as well as off-target effects. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0256DOI Listing
January 2018

Human TCR-MHC coevolution after divergence from mice includes increased nontemplate-encoded CDR3 diversity.

J Exp Med 2017 Nov 23;214(11):3417-3433. Epub 2017 Aug 23.

Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

For thymic selection and responses to pathogens, T cells interact through their αβ T cell receptor (TCR) with peptide-major histocompatibility complex (MHC) molecules on antigen-presenting cells. How the diverse TCRs interact with a multitude of MHC molecules is unresolved. It is also unclear how humans generate larger TCR repertoires than mice do. We compared the TCR repertoire of CD4 T cells selected from a single mouse or human MHC class II (MHC II) in mice containing the human TCR gene loci. Human MHC II yielded greater thymic output and a more diverse TCR repertoire. The complementarity determining region 3 (CDR3) length adjusted for different inherent V-segment affinities to MHC II. Humans evolved with greater nontemplate-encoded CDR3 diversity than did mice. Our data, which demonstrate human TCR-MHC coevolution after divergence from rodents, explain the greater T cell diversity in humans and suggest a mechanism for ensuring that any V-J gene combination can be selected by a single MHC II.
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http://dx.doi.org/10.1084/jem.20161784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679170PMC
November 2017

The immune system prevents recurrence of transplanted but not autochthonous antigenic tumors after oncogene inactivation therapy.

Int J Cancer 2017 12 31;141(12):2551-2561. Epub 2017 Aug 31.

Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

Targeted oncogene inactivation by small molecule inhibitors can be very effective but tumor recurrence is a frequent problem in the clinic. Therapy by inactivation of the cancer-driving oncogene in transplanted tumors was shown to be augmented in the presence of T cells. However, these experiments did not take into account the long-term, usually tolerogenic, interaction of de novo malignancies with the immune system. Here, we employed mice, in which SV40 large T (Tag) and firefly luciferase (Luc) as fusion protein (TagLuc) could be regulated with the Tet-on system and upon activation resulted in tumors after a long latency. TagLuc inactivation induced profound tumor regression, demonstrating sustained oncogene addiction. While tumor relapse after TagLuc inactivation was prevented in immunocompetent mice bearing transplanted tumors, autochthonous tumors relapsed or recurred after therapy discontinuation indicating that the immune system that coevolved with the malignancy over an extended period of time lost the potency to mount an efficient anti-tumor immune response. By contrast, adoptively transferred CD8 T cells targeting the cancer-driving oncogene eradicated recurrent autochthonous tumors, highlighting a suitable therapy option in a clinically relevant model.
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http://dx.doi.org/10.1002/ijc.31009DOI Listing
December 2017

CTLA4 Promotes Tyk2-STAT3-Dependent B-cell Oncogenicity.

Cancer Res 2017 09 17;77(18):5118-5128. Epub 2017 Jul 17.

Department of Onco-Immunology, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, California.

CTL-associated antigen 4 (CTLA4) is a well-established immune checkpoint for antitumor immune responses. The protumorigenic function of CTLA4 is believed to be limited to T-cell inhibition by countering the activity of the T-cell costimulating receptor CD28. However, as we demonstrate here, there are two additional roles for CTLA4 in cancer, including via CTLA4 overexpression in diverse B-cell lymphomas and in melanoma-associated B cells. CTLA4-CD86 ligation recruited and activated the JAK family member Tyk2, resulting in STAT3 activation and expression of genes critical for cancer immunosuppression and tumor growth and survival. CTLA4 activation resulted in lymphoma cell proliferation and tumor growth, whereas silencing or antibody-blockade of CTLA4 in B-cell lymphoma tumor cells in the absence of T cells inhibits tumor growth. This inhibition was accompanied by reduction of Tyk2/STAT3 activity, tumor cell proliferation, and induction of tumor cell apoptosis. The CTLA4-Tyk2-STAT3 signal pathway was also active in tumor-associated nonmalignant B cells in mouse models of melanoma and lymphoma. Overall, our results show how CTLA4-induced immune suppression occurs primarily via an intrinsic STAT3 pathway and that CTLA4 is critical for B-cell lymphoma proliferation and survival. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600851PMC
September 2017

T-cell Receptors for Clinical Therapy: Assessment of Toxicity Risk.

Clin Cancer Res 2017 Oct 23;23(20):6012-6020. Epub 2017 Jun 23.

Laboratory of Tumor Immunology, Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands.

Adoptive therapy with T-cell receptor (TCR)-engineered T cells has shown promising results in the treatment of patients with tumors, and the number of TCRs amenable for clinical testing is expanding rapidly. Notably, adoptive therapy with T cells is challenged by treatment-related side effects, which calls for cautious selection of target antigens and TCRs that goes beyond their mere ability to induce high T-cell reactivity. Here, we propose a sequence of assays to improve selection of TCRs and exemplify risk assessments of on-target as well as off-target toxicities using TCRs directed against cancer germline antigens. The proposed panel of assays covers parameters considered key to safety, such as expression of target antigen in healthy tissues, determination of a TCR's recognition motif toward its cognate peptide, and a TCR's cross-reactivity toward noncognate peptides. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-1012DOI Listing
October 2017

Prevalence of human papillomavirus infection of the anal canal in women: A prospective analysis of high-risk populations.

Oncol Lett 2017 Apr 10;13(4):2495-2501. Epub 2017 Feb 10.

Department of Gynecology and Obstetrics, Ludwig-Maximilian University of Munich, D-80337 Munich, Germany.

Infection with certain types of human papillomavirus (HPV) has been associated with the development of cervical and anal cancer. Worldwide, the incidence of anal cancer has increased markedly. The present study aimed to evaluate the prevalence of HPV infection of the uterine cervix and anal canal in human immunodeficiency virus (HIV)- and non-HIV-infected risk populations. Cervical and anal HPV swabs and cytology samples were collected from 287 patients at the University Hospital of Munich, Germany between 2011 and 2013. Patients were divided into HIV-negative controls (G1) and two risk groups, including HIV-negative patients with cytological abnormalities of the cervix (G2) and HIV-infected patients (G3). Data, including clinical parameters, were analysed. The risk groups had significantly more positive results for HPV in the anus (71.03 and 83.15% for G2 and G3, respectively), as compared with G1. The predominant HPV genotypes found in the anus were high-risk HPV genotypes, which were significantly correlated with concomittant cervical HPV findings. In the risk groups, a significant association between the cytological findings and HPV detection in the cervix was found, while the results of the anus revealed no significance. The results of the present study suggested that the prevalence of HPV infection in the anal canal of risk populations is high. Furthermore, patients with abnormal cervical cytology results and HIV-infected women, irrespective of their individual cervical findings, may have a risk of concomittant anal high-risk HPV infection. Based on the predominant HPV genotypes found in the study, HPV vaccination could reduce the incidence of anal cancer. Nevertheless, high-risk patients should be intensively screened for anal squamous intraepithelial abnormalities to avoid invasive cancer stages.
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http://dx.doi.org/10.3892/ol.2017.5714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403543PMC
April 2017

Tumour ischaemia by interferon-γ resembles physiological blood vessel regression.

Nature 2017 05 26;545(7652):98-102. Epub 2017 Apr 26.

Institute of Immunology, Charité Campus Buch, 13125 Berlin, Germany.

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ-GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.
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http://dx.doi.org/10.1038/nature22311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567674PMC
May 2017

Tumor-Infiltrating Merkel Cell Polyomavirus-Specific T Cells Are Diverse and Associated with Improved Patient Survival.

Cancer Immunol Res 2017 02 16;5(2):137-147. Epub 2017 Jan 16.

Dermatology/Medicine/Pathology, University of Washington, Seattle, Washington.

Tumor-infiltrating CD8 T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8 T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, "KLL") and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer CD8 T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry. TCRβ (TRB) sequencing was performed on tetramer cells from PBMCs or TILs (n = 14) and matched tumors (n = 12). Functional avidity of T-cell clones was determined by IFNγ production. We identified KLL tetramer T cells in 14% of PBMC and 21% of TIL from MCC patients. TRB repertoires were strikingly diverse (397 unique TRBs were identified from 12 patients) and mostly private (only one TCRb clonotype shared between two patients). An increased fraction of KLL-specific TIL (>1.9%) was associated with significantly increased MCC-specific survival P = 0.0009). T-cell cloning from four patients identified 42 distinct KLL-specific TCRa/b pairs. T-cell clones from patients with improved MCC-specific outcomes were more avid (P < 0.05) and recognized an HLA-appropriate MCC cell line. T cells specific for a single MCPyV epitope display marked TCR diversity within and between patients. Intratumoral infiltration by MCPyV-specific T cells was associated with significantly improved MCC-specific survival, suggesting that augmenting the number or avidity of virus-specific T cells may have therapeutic benefit. Cancer Immunol Res; 5(2); 137-47. ©2017 AACR.
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http://dx.doi.org/10.1158/2326-6066.CIR-16-0210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421625PMC
February 2017

Preventing tumor escape by targeting a post-proteasomal trimming independent epitope.

J Exp Med 2016 10 3;213(11):2333-2348. Epub 2016 Oct 3.

Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany

Adoptive T cell therapy (ATT) can achieve regression of large tumors in mice and humans; however, tumors frequently recur. High target peptide-major histocompatibility complex-I (pMHC) affinity and T cell receptor (TCR)-pMHC affinity are thought to be critical to preventing relapse. Here, we show that targeting two epitopes of the same antigen in the same cancer cells via monospecific T cells, which have similar pMHC and pMHC-TCR affinity, results in eradication of large, established tumors when targeting the apparently subdominant but not the dominant epitope. Only the escape but not the rejection epitope required postproteasomal trimming, which was regulated by IFN-γ, allowing IFN-γ-unresponsive cancer variants to evade. The data describe a novel immune escape mechanism and better define suitable target epitopes for ATT.
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http://dx.doi.org/10.1084/jem.20160636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068242PMC
October 2016

Interleukin-7 Modulates Anti-Tumor CD8+ T Cell Responses via Its Action on Host Cells.

PLoS One 2016 22;11(7):e0159690. Epub 2016 Jul 22.

Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany.

The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT) of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7) is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+) host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7) therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159690PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4957759PMC
July 2017

Expression of Activin During and After Chemotherapy in Peripheral Blood of Patients with Primary Breast Cancer.

Anticancer Res 2016 May;36(5):2153-9

Department of Gynaecology and Obstetrics, Munich, Germany.

Background/aim: Activins are dimeric glycoproteins that play a significant role in reproduction and in endocrine-active tumors. The aim of this study was to evaluate the potential correlation between the concentration of activins (activin A, activin B, and activin AB) in patients receiving adjuvant chemotherapy for breast cancer.

Patients And Methods: The serum concentration of activins in 30 patients receiving chemotherapy within the German SUCCESS A study was analyzed using different enzyme-linked immunosorbent assays at three time points: After primary surgery, but before chemotherapy; 4 weeks after the end of chemotherapy; and 2 years after chemotherapy during recurrence-free follow-up.

Results: The activin concentration decreased in all patients after chemotherapy. Premenopausal patients had significantly lower concentrations of activin AB during follow-up than postmenopausal women (p=0.037). Thirteen out of 16 premenopausal patients developed chemotherapy-related amenorrhea (CRA) but did not significantly differ in their activin concentrations compared to the other premenopausal women. A positive human epidermal growth factor receptor 2/neu status was associated with a significant reduction of activin AB concentration (p=0.02), and trastuzumab treatment correlated with significantly decreased activin A concentration (p=0.012).

Conclusion: Serial measurements of activin A concentration might be used for monitoring trastuzumab treatment. A sudden increase of activin concentration could be an early indicator of disease recurrence.
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May 2016

Receptor combinations hone T-cell therapy.

Nat Biotechnol 2016 Apr;34(4):389-91

Max-Delbrck-Center for Molecular Medicine, Berlin, Germany, and the Institute of Immunology, Charité Campus Buch, Berlin, Germany.

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http://dx.doi.org/10.1038/nbt.3539DOI Listing
April 2016

Targeting human melanoma neoantigens by T cell receptor gene therapy.

J Clin Invest 2016 Mar 25;126(3):854-8. Epub 2016 Jan 25.

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy.
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http://dx.doi.org/10.1172/JCI83465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767365PMC
March 2016

Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice.

Nat Biotechnol 2015 Apr 16;33(4):402-7. Epub 2015 Mar 16.

1] Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. [2] Institute of Immunology, Charité Campus Buch, Berlin, Germany.

Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8⁺ T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy.
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http://dx.doi.org/10.1038/nbt.3147DOI Listing
April 2015

Targeting cancer-specific mutations by T cell receptor gene therapy.

Curr Opin Immunol 2015 Apr 27;33:112-9. Epub 2015 Feb 27.

Institute of Immunology, Charité Campus Buch, 13125 Berlin, Germany; Department of Pathology, Committee on Immunology and Committee on Cancer Biology, The University of Chicago, Chicago, IL 60637, USA.

The ease of sequencing the cancer genome, identifying all somatic mutations and grafting mutation-specific T cell receptor (TCR) genes into T cells for adoptive transfer allow, for the first time, a truly tumor-specific and effective therapy. Mutation-specific TCR gene therapy might achieve optimal efficacy with least possible toxicity. Recent clinical data confirm the long-standing evidence from experimental cancer models that antigens encoded by the tumor-specific somatic mutations are potentially the best targets for adoptive T cell therapy. Open questions are, how many somatic mutations create suitable epitopes, whether only individual-specific or also recurrent somatic mutations qualify as suitable epitopes and how neoantigen-specific TCRs are most efficiently obtained. Tumor heterogeneity needs to be considered; therefore, it will be important to identify immunogenic driver mutations that occurred early, are essential for cancer cell survival and present in all cancer cells.
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http://dx.doi.org/10.1016/j.coi.2015.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4557613PMC
April 2015