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Presented is an experimental analysis of the stability of transgene expression, the perturbation of endogenous expression and the perturbation of epigenetic organization upon site-directed delivery of transgenes to the CCR5 and AAVS1 loci in human cells. It provides guidelines for optimal cassette design for stable and nonperturbative gene transfer. Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

T-cell avidity is a major determinant of Adoptive T cell therapy (ACT) efficacy for cancer treatment. However, high-avidity tumor-specific T cells can rarely be isolated from cancer patients, highlighting the need for strategies to enhance the cytotoxic capacity of low-avidity cells. Here, we rescue the anti-tumor functions of low-avidity T cells against pancreatic ductal adenocarcinoma (PDAC) by knocking-out TIGIT, a key inhibitory molecule expressed on exhausted CD8 + T cells infiltrating gastrointestinal tumors. We uncover that TIGIT disruption by base editing boosts the intracellular signal transduction derived from a weak T cell receptor (TCR) engagement enforcing cytoskeletal rearrangements, thus increasing T cell avidity and stabilizing the immunological synapse. Accordingly, TIGIT disruption enables low-avidity T cells to exert robust degranulation, comparable to that of high-avidity T cells, and potent and durable anti-tumor capacity in vivo in male mice. These results highlight TIGIT knockout as a potential strategy to enhance low-avidity T cell function and broaden the repertoire of TCR engineered T cells in the treatment of pancreatic cancer and other solid malignancies. Anti-tumor functions of low-avidity T cells are often suboptimal. Here the authors show that genetic disruption of TIGIT in TCR-engineered T cells enhances their anti-tumor activity against pancreatic and other gastrointestinal cancers by increasing TCR signal strength.

Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. In a phase I–II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per μL or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17–66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34–127) from transplantation and 23 days (13–42) from infusion. Ten patients developed acute GVHD (grade I–IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25–73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. MolMed SpA, Italian Association for Cancer Research.

In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCR ) and permanently disrupted , or genes (IR ) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCR -IR and IR competent (TCR -IR ) cells were tested in short-term co-culture assays and under a chronic stimulation setting . Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. We show that upon chronic stimulation, TCR -IR cells are superior to TCR -IR cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge . Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo, with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

Engineered T cells expressing a tumor antigen specific T cell receptor (TCR) have shown promise for cancer immunotherapy. However, the introduced TCR chains can pair with the endogenous TCR chains in T cells, and in mice, these mismatched TCRs can cause a lethal autoimmune reaction. Provasi et al . now show that they can eliminate expression of the endogenous TCR chains using zinc finger nucleases and express only the desired exogenous TCR by lentiviral transduction. The resultant TCR-edited lymphocytes showed tumor specificity without the risk of off-target toxicity. The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo , thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

Introduction/BackgroundThe presence of an immune-reactive tumor microenvironment in epithelial ovarian cancer (EOC) has boosted the development of immunotherapeutic strategy, that unfortunately have shown limited clinical efficacy. Our aim is to investigate the possible immunomodulatory effect of Olaparib and Trabectedin and whether such treatments might impact on the activity of tumor-specific T cells.MethodologyIn vitro dose finding assays for Olaparib and Trabectedin were conducted on 2 EOC cell lines and 3 patient-derived primary cultures by Incucyte® live cell imaging and flow cytometry (FC). The expression of inhibitory receptor (IR) ligands, activation molecules and tumor associated antigens (TAAs) was evaluated by FC after treatments with sub-optimal doses of each drug. The combined effect of drug treatment and T cells harbouring engineered tumor-specific T-cell receptors (TCRs) was also assessed in co-culture assays on both EOC cell lines and EOC primary cultures.ResultsAfter 24-hours treatment of EOC cells with Olaparib and Trabectedin, we found a significant modulation of some IR ligands such as PD-L1, PD-L2 and CD48 and of MHC class-I molecules. Drug treatments significantly modulated the expression of NY-ESO-1, MUC-1 and WT-1 TAAs. Furthermore, 24-hours pre-treatment with each drug before co-culture with tumor-specific TCR-edited T lymphocytes increased tumor cell death on both EOC cells and primary cultures.ConclusionOur data show that Olaparib and Trabectedin modulate the expression of immune-related molecules on EOC cells, and this effect is associated with an increased killing by tumor-specific TCR-edited T cells. Biological mechanisms at the basis of these findings deserve further investigation.DisclosuresNone

Introduction/BackgroundThe observed clinical benefit of immunotherapy in EOC is limited. During cancer progression, tumor-specific T cells display increased expression of inhibitory receptors (IRs), causing their functional exhaustion. We hypothesize that protecting tumor-specific T cells from immunosuppression by gene editing approaches aimed at reducing IRs expression might maximize their therapeutic potential.MethodologyWe characterized by flow cytometry EOC cell lines and patients’ ascites-derived primary cultures (PC) to evaluate their expression of selected tumor-associated antigens and IR ligands. Considering antigen expression, we selected a HLA-A*02:01-restricted WT1-specific TCR from our library (HD1-TCR) and tested different T-cell products engineered to express HD1-TCR and also harboring gene disruption ( HD1-IRKO) of distinct IRs (2B4, TIM3, LAG3).To functionally characterize the putative advantage provided by IR disruption on the anti-tumor efficacy of engineered T cells, HD1-TCR and HD1-IRKO gene-edited T cells were challenged with WT-1pos HLA-A*02:01pos (or HLA-A*02:01neg, as control) cancer cell lines, PC and tumor derived organoids (PDOs). We evaluated cytokine secretion, T cell activation and apoptosis.ResultsWe observed high expression of WT-1 while IR ligands (IR-L) were expressed by a low percentage of tumor cells and increased by IFNγ exposure. After co-culture with tumor cells, the different T-cell products displayed similar killing of cell lines, while increased killing was observed with HD1-IRKO cells against EOC primary cultures expressing IR-Ls. In particular, TIM-3KO HD1-TCR edited T cells displayed more potent killing of PC expressing TIM-3 IR ligands compared to the other T cell products. Additionally, upon co-culture with HLA-matched PDOs, both TIM-3KO and LAG-3KO HD1-TCR T cells showed an increased induction of cancer cell apoptosis compared to IR-competent cells.ConclusionHD1-IRKO T-cell products appear superior to HD1-TCR IR-competent counterparts in killing WT-1pos HLA-A*02:01 cancer cells. The most consistent advantages were observed with TIM-3KO HD1-TCR.DisclosuresReceipt of grants/research supports from Intellia Therapeutics.Receipt of grants/research supports from GSK, MSD/Astrazeneca, Pharmamar

Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (TCM) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC–expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC–expanded T cells modified with a suicide gene in the setting of haplo-HSCT.