Design and Evaluation of TIM-3-CD28 Checkpoint Fusion Proteins to Improve Anti-CD19 CAR T-Cell Function

Therapeutic targeting of inhibitory checkpoint molecules in combination with chimeric antigen receptor (CAR) T cells is currently investigated in a variety of clinical studies for treatment of hematologic and solid malignancies. However, the impact of co-inhibitory axes and their therapeutic implication remains understudied for the majority of acute leukemias due to their low immunogenicity/mutational load. The inhibitory exhaustion molecule TIM-3 is an important marker for the interaction of T cells with leukemic cells. Moreover, inhibitory signals from malignant cells could be transformed into stimulatory signals by synthetic fusion molecules with extracellular inhibitory receptors fused to an intracellular stimulatory domain. Here, we designed a variety of different TIM-3-CD28 fusion proteins to turn inhibitory signals derived by TIM-3 engagement into T-cell activation through CD28. In the absence of anti-CD19 CAR, two TIM-3-CD28 fusion receptors with large parts of CD28 showed strongest responses in terms of cytokine secretion and proliferation upon stimulation with anti-CD3 antibodies compared to controls. We then combined these two novel TIM-3-CD28 fusion proteins with first- and second-generation anti-CD19 CAR T cells and found that the fusion receptor can increase proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR T cells. These additionally armed CAR T cells showed excellent effector function. In terms of safety considerations, the fusion receptors showed exclusively increased cytokine release, when the CAR target CD19 was present. We conclude that combining checkpoint fusion proteins with anti-CD19 CARs has the potential to increase T-cell proliferation capacity with the intention to overcome inhibitory signals during the response against malignant cells

Genome‐wide off‐target analyses of CRISPR/Cas9‐mediated T‐cell receptor engineering in primary human T cells

Objectives Exploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T-cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off-the-shelf options to emerging adoptive T-cell transfer (ACT) approaches. Targeted CRISPR/Cas9-mediated double-strand breaks in the DNA enable knockout or knock-in engineering. Methods Here, we perform CRISPR/Cas9-mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T-cell products. Whole-genome sequencing was performed to analyse whether CRISPR/Cas9-mediated DNA double-strand break at the TCR locus is associated with off-target events in human primary T cells. Results TCRα chain and TCRβ chain knockout leads to high on-target InDel frequency and functional knockout. None of the predicted off-target sites could be confirmed experimentally, whereas whole-genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low-frequency off-target events genome-wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low-frequency InDels. Therefore, off-target events are unlikely to be caused by the CRISPR/Cas9 engineering. Conclusion The combinatorial approach of whole-genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9-mediated TCR knockout without potentially harmful exonic off-target effects

Functional characterization of TRIP6 in Ewing's Sarcoma

Das Ewing-Sarkom (EFT) ist nach dem Osteosarkom das zweihäufigste Knochen-assoziierte Malignom im Kindesalter. Das entscheidende Ereignis in der Pathogenese dieser Entität stellt eine chromosomale Translokation dar, welche zur Entstehung eines chimären Transkriptionsfaktors, meist EWS-FLI1, führt. Unsere Absicht war es, die Mechanismen zu verstehen, die letztlich zur Metastasierung von Ewing-Sarkomen mit der damit verbundenen, infausten Prognose führen. Die Mitglieder der Zyxin-Proteinfamilie sind in vielfältige zelluläre Funktionen involviert. Hierbei nehmen sie, teilweise funktionell redundant, Einfluss auf zytoplasmatische und nukleäre Prozesse. Durch Analyse von öffentlich verfügbaren Microarraydaten konnten wir belegen, dass lediglich das Protein TRIP6 (thyroid receptor interacting protein 6) aus der Familie in EFT deutlich überexprimiert ist. Dieses Protein ist, neben seiner Funktion in der Organisation des Zytoskeletts, auch nukleär als Kotranskriptionsfaktor und als Element der Telomerprotektion tätig. Vielfach wurde eine Implikation des multifunktionellen Adaptorproteins in maligne Prozesse dokumentiert. Die Überexpression von TRIP6 in EFT ist jedoch unabhängig von EWS-FLI1. Eine Bindung von EWS-FLI1 an eine putative Bindungsstelle im Promotor von TRIP6 konnte nicht nachgewiesen werden. Die Analyse von Microarrays nach TRIP6-Knockdown in EFT-Zelllinien identifizierte mehrere Gensets, welche mit Proliferation und Invasivität assoziiert sind und die nach TRIP6-Knockdown vermindert exprimiert werden. Die für Malignome pathogenetisch relevanten Zielgene Radixin, CD164 und CRYZ konnten als Zielgene des Kotranskriptionsfaktors TRIP6 durch qRT-PCR und Western Blot bestätigt werden. Durch RNA-Interferenz-mediierte Verminderung der Proteinmenge von TRIP6 in EFT kam es zu einer deutlich reduzierten Klonogenität und Migration der Zellen in vitro. Nach induzierbarem TRIP6-Knockdown konnte eine verminderte Tumorigenität und hepatische Metastasierung von hierfür generierten EFT-Einzelzellklonen in vivo beobachtet werden. Zusammengefasst deuten diese Daten auf eine Rolle von TRIP6 in der Pathogenese der EFT und insbesondere beim Prozess der Metastasierung hin. Somit legen diese Ergebnisse eine weitere Evaluierung von TRIP6 als Biomarker oder molekulare Zielstruktur für therapeutische Ansätze in EFT nahe.Ewing’s sarcoma (ES) is the second most common bone-associated malignancy in children that is driven by the fusion oncogene EWS/FLI1. Despite the great propensity of ES toward early metastasis, recent evidence showed that EWS/FLI1 expression reduces migration and invasiveness of ES cells. However, we strived for exploring the underlying mechanisms of how ES maintains its basal migratory and invasive properties ultimately contributing to metastasis. We focused on the Zyxin-protein family comprising key players in actin remodeling, migration, and invasion. By interrogation of published microarray data, we observed that of all seven Zyxin-proteins only TRIP6 (thyroid receptor interacting protein 6) is highly overexpressed in ES. Besides its effects on cytoskeletal organization, TRIP6 is a nucleocytoplasmic shuttling protein involved in telomere protection, apoptosis, chemo-resistance, and transcriptional control. Initial experiments indicate that TRIP6 expression is independent of EWS/FLI1. However, RNAi-mediated knockdown of TRIP6 in ES cells significantly reduced clonogenicity and migration, whereas proliferation and adhesion were not reduced. DNA microarrays revealed that TRIP6 knockdown is accompanied by the downreguation of important pro-migratory and pro-invasive genes such as Radixin, CD164, and crystalline zeta.Taken together, these data indicate that TRIP6 might partially account for the EWS/FLI1-autonomous migratory and invasive properties of E

Strategies of adoptive T -cell transfer to treat refractory viral infections post allogeneic stem cell transplantation

Abstract Background Allogeneic hematopoietic stem cell transplantation (HSCT) can expose patients to a transient but marked immunosuppression, during which viral infections are an important cause of morbidity and mortality. Adoptive transfer of virus-specific T cells is an attractive approach to restore protective T -cell immunity in patients with refractory viral infections after allogeneic HSCT. Objectives This narrative review summarizes clinical evidence and developments of almost 30 years of adoptive T -cell transfer. The review is based on evidence extracted from PubMed searches and the clinical and experimental work of the authors. Content Viral infections after HSCT are frequently caused by the endogenous reactivation of persistent pathogens such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV). Current antiviral medication is not satisfactory and does not treat the underlying pathophysiology which is the lack of specific T -cell immunity. Adoptive transfer of virus-specific T cells could be a potentially curative, pathogen-specific, and non-toxic treatment providing long-term immunity against the virus. The isolation of virus-specific T cells from a healthy donor and infusion into a recipient is known as adoptive T -cell transfer and has been performed in many patients using different treatment protocols. Based on basic research, new isolation protocols aim at a safe and fast availability of cellular products for adoptive T -cell transfer. We summarize preclinical and clinical data on each of the main pathogens and on the technical approaches currently available to target either single antigens or even multiple pathogens. Conclusion Cellular therapy is considered as one of the major recent breakthroughs in medicine. Translation of this individualized treatment into first-line clinical routine is still limited. Main hurdles are availability of the technique, limited compatibility of classical phase III designs with cellular therapy, and regulatory restrictions. Multinational efforts are required to clarify the status of cellular treatment in first-line clinical routine with the overall objective to strengthen evidence-based treatment guidelines for the treatment of refractory viral infections post HSCT

Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing

International audienceCircular RNA (circRNA) is a noncoding RNA class with important implications for gene expression regulation, mostly by interaction with other RNA species or RNA-binding proteins. While the commonly applied short-read Illumina RNA-sequencing techniques can be used to detect circRNAs, their full sequence is not revealed. However, the complete sequence information is needed to analyze potential interactions and thus the mechanism of action of circRNAs. Here, we present an improved protocol to enrich and sequence full-length circRNAs by using the Oxford Nanopore long-read sequencing platform. The protocol involves an enrichment of lowly abundant circRNAs by exonuclease treatment and negative selection of linear RNAs. Then, a cDNA library is created and amplified by PCR. This protocol provides enough material for several sequencing runs. The library is used as input for ligation-based sequencing together with native barcoding. Stringent quality control of the libraries is ensured by a combination of Qubit, Fragment Analyzer and qRT-PCR. Multiplexing of up to 4 libraries yields in total more than 1–2 Million reads per library, of which 1–2% are circRNA-specific reads with >99% of them full-length. The protocol works well with human cancer cell lines. We further provide suggestions for the bioinformatic analysis of the created data, as well as the limitations of our approach together with recommendations for troubleshooting and interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a noncoding RNA type involved in a growing number of physiologic and pathologic conditions. Metadata Associated content

Antiemetic Prophylaxis with Fosaprepitant and 5-HT3-Receptor Antagonists in Pediatric Patients Undergoing Autologous Hematopoietic Stem Cell Transplantation

Background: High-dose myeloablative conditioning prior to autologous hematopoietic stem cell transplantation (autoHSCT) in pediatric patients is usually highly emetogenic. The antiemetic neurokinin-1 receptor antagonist fosaprepitant was safe and effective in children receiving highly emetogenic chemotherapy. Data on fosaprepitant during autoHSCT in children are currently not available. Methods: A total of 35 consecutive pediatric patients, who received an antiemetic prophylaxis with fosaprepitant (4 mg/kg;single dose, max. 1 x 150 mg/kg BW) and ondansetron (24-hours continuous infusion;8-32 mg/24h) or granisetron (2 x 40 mu g/kg.d(-1)) during highly emetogenic conditioning chemotherapy before autoHSCT were retrospectively analyzed, and their results were compared with a control group comprising 35 consecutive pediatric patients, who received granisetron or ondansetron only. The antiemetic efficacy and the safety of the two prophylaxis regimens were compared with respect to three time periods after the first chemotherapy administration (0-24h, >24-120h, >120-240h). Results: Clinical adverse events and clinically relevant increases/decreases of laboratory markers were similarly low and did not significantly differ between the two study groups (p>0.05). The registered number of vomiting events was significantly higher in the control group in the time periods of 0-24h (64 vs 22 events;p24-120h (135 vs 78 events;p120-240h (268 vs 105 events;p24-120h (100% vs 74.3%) but not the other analyzed time periods (p>0.05). Conclusion: The fosaprepitant-based antiemetic prophylaxis was safe, well tolerated and significantly reduced vomiting in children undergoing highly emetogenic chemotherapy prior to autoHSCT. Prospective randomized trials are necessary to confirm these results

Leukemia escape in immune desert: intraocular relapse of pediatric pro-B-ALL during systemic control by CD19-CAR T cells

Background Relapsed/refractory B-precursor acute lymphoblastic leukemia (BCP-ALL) remains a major therapeutic challenge in pediatric hematology. Chimeric antigen receptor (CAR) T cells targeting CD19 have shown remarkable initial response rates in BCP-ALL patients, while long-term leukemia control rate is only about 50%. So far, main mechanisms of BCP-ALL relapse after CD19-CAR T-cell therapy have been either insufficient CAR T-cell persistence in vivo or loss of surface CD19.Case Report Here, we report an exceptional presentation of BCP-ALL relapse in the eye during the systemic control through CAR T-cell therapy. We report a case of fatal intraocular relapse in a pediatric patient with pro-B-ALL after initial response to CD19-CAR T-cell therapy. One month after CD19-CAR T-cell therapy, remission was documented by bone marrow aspirate analysis with absence of CD19+ cells and CD19-CAR T cells could be detected in both peripheral blood and bone marrow. At the same time, however, the patient presented with progressive visual disturbance and CD19+ cells were found within the anterior chamber of the eye. Despite local and systemic therapy, ocular relapse led to BCP-ALL dissemination and systemic relapse within weeks. The eye represents a rare site for local manifestation of BCP-ALL, but isolated intraocular relapse is a clinically unreckoned presentation of BCP-ALL in the era of CD19-CAR T cells.Conclusion During systemic control of BCP-ALL through CD19-CAR T cells, relapse can emerge in the eye as an immune-privileged organ. Ocular symptoms after CD19-CAR T-cell therapy should guide the clinician to elucidate the etiology in a timely fashion in order to adjust leukemia treatment strategy. Both, local immune escape as well as insufficient CAR T-cell persistence may have contributed to relapse in the reported patient. Mechanisms of relapse in an immune desert under CAR T-cell therapy require future clinical and experimental attention. In particular, ocular symptoms after CAR T-cell therapy should be considered a potentially early sign of leukemia relapse

Antiemetic Prophylaxis with Fosaprepitant and 5-HT3-Receptor Antagonists in Pediatric Patients Undergoing Autologous Hematopoietic Stem Cell Transplantation

Background: High-dose myeloablative conditioning prior to autologous hematopoietic stem cell transplantation (autoHSCT) in pediatric patients is usually highly emetogenic. The antiemetic neurokinin-1 receptor antagonist fosaprepitant was safe and effective in children receiving highly emetogenic chemotherapy. Data on fosaprepitant during autoHSCT in children are currently not available. Methods: A total of 35 consecutive pediatric patients, who received an antiemetic prophylaxis with fosaprepitant (4 mg/kg;single dose, max. 1 x 150 mg/kg BW) and ondansetron (24-hours continuous infusion;8-32 mg/24h) or granisetron (2 x 40 mu g/kg.d(-1)) during highly emetogenic conditioning chemotherapy before autoHSCT were retrospectively analyzed, and their results were compared with a control group comprising 35 consecutive pediatric patients, who received granisetron or ondansetron only. The antiemetic efficacy and the safety of the two prophylaxis regimens were compared with respect to three time periods after the first chemotherapy administration (0-24h, >24-120h, >120-240h). Results: Clinical adverse events and clinically relevant increases/decreases of laboratory markers were similarly low and did not significantly differ between the two study groups (p>0.05). The registered number of vomiting events was significantly higher in the control group in the time periods of 0-24h (64 vs 22 events;p24-120h (135 vs 78 events;p120-240h (268 vs 105 events;p24-120h (100% vs 74.3%) but not the other analyzed time periods (p>0.05). Conclusion: The fosaprepitant-based antiemetic prophylaxis was safe, well tolerated and significantly reduced vomiting in children undergoing highly emetogenic chemotherapy prior to autoHSCT. Prospective randomized trials are necessary to confirm these results

Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. T cell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive T cell transfer (ACT) approaches. T cell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific T cells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated ex vivo and preclinically engineered into the TCR locus of allogeneic third-party primary T cells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited T cells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, ex vivo isolated TCRs with clinical proven protection through ACT could be redirected into T cells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections