Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

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.

CRISPR activation and interference screens decode stimulation responses in primary human T cells.

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.

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.

Robust T cell activation requires an eIF3-driven burst in T cell receptor translation.

Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here, we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3'-untranslated regions (3'-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3'-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering.

As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.

Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

CLEC12A and CD33 coexpression as a preferential target for pediatric AML combinatorial immunotherapy.

Emerging immunotherapies such as chimeric antigen receptor T cells have advanced the treatment of acute lymphoblastic leukemia. In contrast, long-term control of acute myeloid leukemia (AML) cannot be achieved by single lineage-specific targeting while sparing benign hematopoiesis. In addition, heterogeneity of AML warrants combinatorial targeting, and several suitable immunotargets (HAVCR2/CD33 and HAVCR2/CLEC12A) have been identified in adult AML. However, clinical and biologic characteristics of AML differ between children and the elderly. Here, we analyzed 36 bone marrow (BM) samples of pediatric AML patients and 13 age-matched healthy donors using whole RNA sequencing of sorted CD45dim and CD34+CD38-CD45dim BM populations and flow cytometry for surface expression of putative target antigens. Pediatric AML clusters apart from healthy myeloid BM precursors in principal-component analysis. Known immunotargets of adult AML, such as IL3RA, were not overexpressed in pediatric AML compared with healthy precursors by RNA sequencing. CD33 and CLEC12A were the most upregulated immunotargets on the RNA level and showed the highest surface expression on AML detected by flow cytometry. KMT2A-mutated infant AML clusters separately by RNA sequencing and overexpresses FLT3, and hence, CD33/FLT3 cotargeting is an additional specific option for this subgroup. CLEC12A and CD33/CLEC12Adouble-positive expression was absent in CD34+CD38-CD45RA-CD90+ hematopoietic stem cells (HSCs) and nonhematopoietic tissue, while CD33 and FLT3 are expressed on HSCs. In summary, we show that expression of immunotargets in pediatric AML differs from known expression profiles in adult AML. We identify CLEC12A and CD33 as preferential generic combinatorial immunotargets in pediatric AML and CD33 and FLT3 as immunotargets specific for KMT2A-mutated infant AML.

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.

Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR).

The chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, nor actin remodeling following CAR activation. However, CAR T cells still require LAT for an optimal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.

CRISPR-Cas9-Mediated Glucocorticoid Resistance in Virus-Specific T Cells for Adoptive T Cell Therapy Posttransplantation.

Immunosuppression posttransplantation exposes patients to an increased risk for refractory viral infections as an important cause of morbidity and mortality. Protective T cell immunity can be restored by adoptive T cell transfer, but ongoing immunosuppression limits efficacy of T cell responses. In order to deliver protection against viral pathogens and allow at the same time necessary steroid therapy, we generated glucocorticoid-resistant T cells by CRISPR-Cas9-mediated knockout of the glucocorticoid receptor in primary human virus-specific T cell products. Characterization of the T cell product revealed high efficiency of glucocorticoid receptor knockout and high purity of virus-specific T cells. This tandem T cell engineering preserved protective T cell functionality, such as cytotoxicity, CD107a degranulation, proliferative capacity, and cytokine release patterns. Virus-specific T cells with glucocorticoid receptor knockout were resistant to the suppressive effect of dexamethasone treatment on lymphocyte proliferation and cytokine secretion (tumor necrosis factor alpha [TNF-α], interleukin-4 [IL-4], IL-6, and sFas). Additionally, glucocorticoid receptor knockout cells remained sensitive to cyclosporine A treatment, thereby providing a rescue approach for patients in case of safety issues. This novel approach provides a therapeutic option for the treatment of patients with viral infections after transplantation who are receiving glucocorticoid therapy.

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR.

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRß chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.

Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.

Leukemia-induced dysfunctional TIM-3+CD4+ bone marrow T cells increase risk of relapse in pediatric B-precursor ALL patients.

Interaction of malignancies with tissue-specific immune cells has gained interest for prognosis and intervention of emerging immunotherapies. We analyzed bone marrow T cells (bmT) as tumor-infiltrating lymphocytes in pediatric precursor-B cell acute lymphoblastic leukemia (ALL). Based on data from 100 patients, we show that ALL is associated with late-stage CD4+ phenotype and loss of early CD8+ T cells. The inhibitory exhaustion marker TIM-3 on CD4+ bmT increased relapse risk (RFS = 94.6/70.3%) confirmed by multivariate analysis. The hazard ratio of TIM-3 expression nearly reached the hazard ratio of MRD (7.1 vs. 8.0) indicating that patients with a high frequency of TIM-3+CD4+ bone marrow T cells at initial diagnosis have a 7.1-fold increased risk to develop ALL relapse. Comparison of wild type primary T cells to CRISPR/Cas9-mediated TIM-3 knockout and TIM-3 overexpression confirmed the negative effect of TIM-3 on T cell responses against ALL. TIM-3+CD4+ bmT are increased in ALL overexpressing CD200, that leads to dysfunctional antileukemic T cell responses. In conclusion, TIM-3-mediated interaction between bmT and leukemia cells is shown as a strong risk factor for relapse in pediatric B-lineage ALL. CD200/TIM-3-signaling, rather than PD-1/PD-L1, is uncovered as a mechanism of T cell dysfunction in ALL with major implication for future immunotherapies.

Efficacy, safety and feasibility of fosaprepitant for the prevention of chemotherapy-induced nausea and vomiting in pediatric patients receiving moderately and highly emetogenic chemotherapy - results of a non-interventional observation study.

BACKGROUND: Chemotherapy-induced nausea and vomiting (CINV) belong among the most burdensome side effects in hemato-oncology. Mostly, a combination of ondansetron and dexamethasone is used as antiemetic prophylaxis in pediatric patients undergoing emetogenic chemotherapy. However, dexamethasone is prohibited in different pediatric chemotherapy protocols. Currently, data on the use of ondansetron with the new antiemetic agent fosaprepitant without dexamethasone is not available for pediatric patients. METHODS: In this non-interventional observation study, 79 pediatric patients with a median age of 8.0 years (range 0.5-17.9 years) who received a CINV prophylaxis regimen with either fosaprepitant (4 mg/kg; maximum 150 mg) and ondansetron (as 24-h continuous infusion) (n = 40; fosaprepitant group/FG) or ondansetron only (n = 39; control group/CG) during moderately or highly emetogenic chemotherapy were analyzed. The groups were analyzed and compared for frequency of vomiting, administered doses of on-demand antiemetic dimenhydrinate and adverse events during the acute (0-24 h after chemotherapy administration) and delayed (> 24 h-120 h) CINV phases. RESULTS: A total of 112 and 116 chemotherapy blocks were analyzed in the fosaprepitant and the control group, respectively. The emetogenic potential of the administered chemotherapy did not significantly differ (p = 0.8812) between the two cohorts. In the acute CINV phase, the percentage of patients experiencing vomiting (n = 26 patients) and the vomiting events were significantly higher (p = 0.0005 and p < 0.0001, respectively) in the CG (n = 26 patients (66.7%); 88 events) compared with the FG (n = 10 patients (25.0%); 37 events). In the delayed CINV phase, the percentage of patients experiencing vomiting and the vomiting events were also significantly higher (p = 0.0017 and p < 0.0001, respectively) in the CG (n = 31 patients (79.5%); 164 events) compared with the FG (n = 17 patients (42.5%); 103 events). Additionally, significantly more dimenhydrinate doses were administered in the CG compared with the FG patients (n = 322/n = 198; p < 0.0001). The occurrence of adverse events did not significantly differ between the two groups (p > 0.05). CONCLUSION: Fosaprepitant (4.0 mg/kg) in addition to ondansetron, without application of dexamethasone, was well tolerated, safe, effective and superior to ondansetron only as CINV prophylaxis in pediatric patients during moderately and highly emetogenic chemotherapy.

Low mutational load in pediatric medulloblastoma still translates into neoantigens as targets for specific T-cell immunotherapy.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in childhood and adolescence. Although some patients present with distinct genetic alterations, such as mutated TP53 or MYC amplification, pediatric medulloblastoma is a tumor entity with minimal mutational load and low immunogenicity. METHODS: We identified tumor-specific mutations using next-generation sequencing of medulloblastoma DNA and RNA derived from primary tumor samples from pediatric patients. Tumor-specific mutations were confirmed using deep sequencing and in silico analyses predicted high binding affinity of the neoantigen-derived peptides to the patients' human leukocyte antigen molecules. Tumor-specific peptides were synthesized and used to induce a de novo T-cell response characterized by interferon gamma and tumor necrosis factor alpha release of CD8+ cytotoxic T cells in vitro. RESULTS: Despite low mutational tumor burden, at least two immunogenic tumor-specific peptides were identified in each patient. T cells showed a balanced CD4/CD8 ratio and mostly effector memory phenotype. Induction of a CD8-specific T-cell response was achieved for the neoepitopes derived from Histidine Ammonia-Lyase (HAL), Neuraminidase 2 (NEU2), Proprotein Convertase Subtilisin (PCSK9), Programmed Cell Death 10 (PDCD10), Supervillin (SVIL) and tRNA Splicing Endonuclease Subunit 54 (TSEN54) variants. CONCLUSION: Detection of patient-specific, tumor-derived neoantigens confirms that even in tumors with low mutational load a molecular design of targets for specific T-cell immunotherapy is possible. The identified neoantigens may guide future approaches of adoptive T-cell transfer, transgenic T-cell receptor transfer or tumor vaccination.

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

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.

Systematic identification of cancer-specific MHC-binding peptides with RAVEN.

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Immunogenic peptides encoded by cancer-specific genes (CSGs) may enable targeted immunotherapy, even of oligo-mutated cancers, which lack neo-antigens generated by protein-coding missense mutations. Here, we describe an algorithm and user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues) that automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from transcriptome data. We applied RAVEN to a dataset assembled from 2,678 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on oligo-mutated pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs, and identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated in cancer cell lines and in a comprehensive tissue-microarray. Subsequently, RAVEN identified likely immunogenic CSG-encoded peptides by predicting their affinity to MHCs and excluded sequence identity to abundantly expressed proteins by interrogating the UniProt protein-database. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed binding-kinetics like a very immunogenic influenza control peptide. Collectively, we provide an exquisitely curated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer types, and a freely available software (https://github.com/JSGerke/RAVENsoftware) to easily apply our algorithm to any gene expression dataset. We anticipate that our peptide libraries and software constitute a rich resource to advance anti-cancer immunotherapy.