Ugur Uslu

Autoimmune Colitis and Subsequent CMV-induced Hepatitis After Treatment With Ipilimumab.

Ipilimumab, a humanized CTLA-4 antibody, improves overall survival in patients with metastatic melanoma. However, immune-related adverse effects occur in about 65% of ipilimumab-treated patients and have to be adequately managed. A 55-year-old patient developed grade 3 autoimmune colitis 7 weeks after initiation of ipilimumab treatment and subsequently hepatitis with grade 3 elevation of transaminases and γ-glutamyl transferase. Colitis manifested with up to 18 watery and bloody stools per day and severe attacks of abdominal pain. After exclusion of infectious causes, immunosuppression with corticosteroids was initiated. Because of recurrent abdominal pain, spontaneous perforation of the colon had to be excluded. Elevated liver function tests (grade 3 CTCAE) occurred and differential diagnosis included immune-mediated, toxic, and viral hepatitis. It is interesting to note that, not an immune-mediated but a cytomegalovirus-induced hepatitis was diagnosed by serum blood tests and liver biopsy and was subsequently successfully treated. Careful elaboration of the patient under immunotherapy was essential as further immunosuppression mandatory for autoimmune hepatitis would have worsened the viral hepatitis. In conclusion, cytomegalovirus reactivation should be included in the differential in patients under immunotherapy with checkpoint inhibitors and has to be considered as a cause for morbidity.

RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma

BackgroundAdoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA.MethodsPBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed.ResultsUsing zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells.ConclusionWe present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016).

Combining a chimeric antigen receptor and a conventional T‐cell receptor to generate T cells expressing two additional receptors (TETARs) for a multi‐hit immunotherapy of melanoma

The adoptive transfer of engineered T cells represents an important approach in immunotherapy of melanoma. However, relapse of the tumor can occur due to immune‐escape mechanisms developed by the tumor cells, for example antigen loss, downregulation of the major histocompatibility complex presentation machinery and defects in antigen processing. To counteract these mechanisms, we combined a T‐cell receptor and a chimeric antigen receptor, specific for different common melanoma antigens, gp100 (PMEL) and MCSP (HMW‐MAA), to generate functional CD8+ T cells expressing two additional receptors (TETARs) by electroporation of receptor‐encoding mRNA. These TETARs produced cytokines and were lytic upon recognition of each of their cognate antigens, while no reciprocal inhibition of the receptors occurred. When stimulated with target cells, which express both antigens, an enhanced effect was suggested. The confirmation that chimeric antigen receptors and T‐cell receptors can be functionally combined opens up new avenues in cancer immunotherapy, and the generation of TETARs helps by‐passing major mechanisms by which tumor cells escape immune recognition.

The siRNA-mediated downregulation of PD-1 alone or simultaneously with CTLA-4 shows enhanced in vitro CAR-T-cell functionality for further clinical development towards the potential use in immunotherapy of melanoma

Chimeric antigen receptor (CAR)‐T cells have been used successfully for cancer immunotherapy. While substantial tumor regression was observed in leukaemia and lymphoma, CAR therapy of solid tumors needs further improvement. A major obstacle to the efficiency of engineered T cells is posed by triggering of inhibitory receptors, for example programmed cell death protein 1 (PD‐1) and cytotoxic T lymphocyte–associated protein 4 (CTLA‐4), leading to an impaired antitumor activity. To boost CAR‐T‐cell function, we co‐electroporated T cells with both, mRNA encoding a CAR specific for chondroitin sulphate proteoglycan 4 (CSPG4) and small‐interfering RNAs (siRNAs) to downregulate PD‐1 (siPD‐1) and CTLA‐4 (siCTLA‐4). Flow cytometry revealed that activation‐induced upregulation of both PD‐1 and CTLA‐4 was suppressed when compared to CAR‐T cells electroporated with negative control siRNA. The siRNA transfection showed no influence on CAR expression of engineered T cells. Functionality assays were performed using PD‐L1‐ and CD80‐transfected melanoma cells endogenously expressing CSPG4. CAR‐T cells transfected with siPD‐1 alone showed improvement in cytokine secretion. Additionally, CAR‐T cells transfected with either siPD‐1 alone or together with siCTLA‐4 exhibited a significantly increased cytotoxicity. No or only little effects were observed when CAR‐T cells were co‐transfected with siCTLA‐4 only. Taken together, it is feasible to optimize CAR‐T cells by co‐transfection of CAR‐encoding mRNA and siRNAs to downregulate inhibitory receptors. Our in vitro data indicate an improvement of the functionality of these CAR‐T cells, suggesting that this strategy could represent a novel method to enhance CAR‐T‐cell immunotherapy of cancer.

Automated closed-system manufacturing of human monocyte-derived dendritic cells for cancer immunotherapy

Dendritic cell (DC)-based vaccines have been successfully used for immunotherapy of cancer and infections. A major obstacle is the need for high-level class A cleanroom cGMP facilities for DC generation. The CliniMACS Prodigy® (Prodigy) represents a new platform integrating all GMP-compliant manufacturing steps in a closed system for automated production of various cellular products, notably T cells, NK cells and CD34+ cells. We now systematically tested its suitability for producing human mature monocyte-derived DCs (Mo-DCs), and optimized it by directly comparing the Prodigy approach to our established standard production of Mo-DCs from elutriated monocytes in dishes or bags. Upon step-by-step identification of an optimal cell concentration for the Prodigys CentriCult culture chamber, the total yield (% of input CD14+ monocytes), phenotype, and functionality of mature Mo-DCs were equivalent to those generated by the standard protocol. Technicians labor time was comparable for both methods, but the Prodigy approach significantly reduced hands-on time and high-level clean room resources. In summary, using our optimized conditions for the CliniMACS Prodigy, human Mo-DCs for clinical application can be generated almost automatically in a fully closed system. A significant drawback of the Prodigy approach was, however, that due to the limited size of the CentriCult culture chamber, in contrast to our standard semi-closed elutriation approach, only one fourth of an apheresis could be processed at once.

The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

CAR-T cell therapy in melanoma: a future success story?

Chimeric antigen receptor (CAR)‐T cells are one of the impressive recent success stories of anti‐cancer immunotherapy. Especially in haematological malignancies, this treatment strategy has shown promising results leading to the recent approval of two CAR‐T cell constructs targeting CD19 in the United States and the European Union. After the huge success in haematological cancers, the next step will be the evaluation of its efficacy in different solid tumors, which is currently investigated in preclinical as well as clinical settings. A commonly examined tumor model in the context of immunotherapy is melanoma, since it is known for its immunogenic features. However, the first results of CAR‐T cell therapy in solid tumors did not reveal the same impressive outcomes that were observed in haematological malignancies, as engineered cells need to cope with several challenges. Obstacles include the lack of migration of CAR‐T cells from blood vessels to the tumor site as well as the immunosuppressive tumor microenvironment within solid tumors. Another hurdle is posed by the identification of an ideal target antigen to avoid on‐target/off‐tumor toxicities. Regarding immune escape mechanisms, which can be developed by tumor cells to bypass immune recognition, the observation of antigen loss should also be considered. This article gives an overview of the challenges displayed in CAR‐T cell therapy for the use in solid tumors and discusses different new strategies and approaches that deal with these problems in order to improve CAR‐T cell therapy, particularly for its use in melanoma.

ADF Winter School-An exciting concept of the Arbeitsgemeinschaft Dermatologische Forschung to connect young scientists and clinician scientists in Dermatology at the top of Germany

1Department of Dermatology, University of Tübingen, Tübingen, Germany 2CRC/TRR156 University of Heidelberg, Tübingen and Mainz, Germany 3Department of Dermatology, University of Cologne, Cologne, Germany 4Department of Dermatology, University of Freiburg, Freiburg, Germany 5Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany 6Department of Dermatology, University of Heidelberg, Heidelberg, Germany 7Department of Dermatology, Technical University of Munich, Munich, Germany 8Klinikum Dortmund GmbH, Dortmund, Germany 9University Hospital of Dermatology, University of Salzburg, Salzburg, Austria 10Department of Dermatology, University of Regensburg, Regensburg, Germany 11Center for Dermatology and Immunology, Städtisches Klinikum Dessau, Dessau, Germany 12Department of Dermatology, Medical University of Vienna, Vienna, Austria 13Department of Dermatology, University of Leipzig, Leipzig, Germany 14Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany 15Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland 16Department of Dermatology, University of Münster, Münster, Germany 17Department of Dermatology, University of Düsseldorf, Düsseldorf, Germany 18Department of Dermatology and Allergy, Charité Universitätsmedizin Berlin, Berlin, Germany 19Department of Dermatology, University of Erlangen, Erlangen, Germany 20Department of Immunology, Universitätsspital Zürich, Zürich, Switzerland 21Department of Dermatology, University of Mainz, Mainz, Germany 22CRC1009 and Cells in Motion – Cluster of Excellence, University of Münster, Münster, Germany A D F N E W S

Intracorneal Hematoma Showing Clinical and Dermoscopic Features of Acral Lentiginous Melanoma

Intra- and subcorneal hematoma, a skin alteration seen palmar and plantar after trauma or physical exercise, can be challenging to distinguish from in situ or invasive acral lentiginous melanoma. Thus, careful examination including dermoscopic and histologic assessment may be necessary to make the correct diagnosis. We here present a case of a 67-year-old healthy female patient who presented with a pigmented plantar skin alteration. Differential diagnoses included benign skin lesions, for example, hematoma or melanocytic nevus, and also acral lentiginous melanoma or melanoma in situ. Since clinical and dermoscopic examinations did not rule out a malignant skin lesion, surgical excision was performed and confirmed an intracorneal hematoma. In summary, without adequate physical trigger, it may be clinically and dermoscopically challenging to make the correct diagnosis in pigmented palmar and plantar skin alterations. Thus, biopsy or surgical excision of the skin alteration may be necessary to rule out melanoma.

Randomized, open-label phase III study to evaluate the adjuvant vaccination with tumor RNAloaded autologous dendritic cells versus observation of patients with resected monosomy 3 uveal melanoma

Traumatic isolated sixth cranial nerve palsy is a rare condition that has been reported to be as low as 1% to 2, 7% following traumatic brain injury. The sixth nerve innervates the ipsilateral lateral rectus which abducts the eye. Isolated loss of lateral gaze with no other cranial nerve signs and muscular entrapment is thought to be resulted from an injury to the peripheral nerve along its course from the brain stem to the lateral rectus. We presented a case of traumatic isolated unilateral sixth cranial nerve palsy in a female patient with diplopia and restriction left eye movement to lateral following head trauma after accident. Head Computed Tomography (CT) scan showed left frontal bone fracture involving the lateral wall of the orbit and also left retro orbital hemorrhage with no other lesions noted in the brain. Eye examination revealed isolated sixth cranial nerve palsy with normal vision of both eyes. Here we discussed about the possible mechanism, differential diagnosis and also management of the patient. Department of Ophthalmology, Saiful Anwar Hospital – Brawijaya University, Malang, Indonesia. *Corresponding author Vierlia, Wino Vrieda, Department of Ophthalmology, Saiful Anwar Hospital Brawijaya University, Malang, Indonesia, E-mail: vrieda_v@yahoo.com. Submitted: 09 Feb 2017; Accepted: 02 Mar 2017; Published: 07 Mar 2017