Key Points Genomic disruption of CD7 prior to CAR transduction allows generation of CD7 CAR T cells without extensive self-antigen-driven fratricide. CD7 CAR T cells have robust activity against T-cell malignancies in vitro and in vivo.

SUMMARY Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time-consuming and expensive. Here we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs, or Cas9-guide-RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (~75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.

The field of gene therapy has been galvanized by the discovery of the highly efficient and adaptable site-specific nuclease system CRISPR/Cas9 from bacteria. 1,2 Immunity against therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative treatment and may pose significant safety issues. [3][4][5] Immunocompetent mice treated with CRISPR/Cas9-encoding vectors exhibit humoral and cellular immune responses against the Cas9 protein, that impact the efficacy of treatment and can cause tissue damage. 5,6 Most applications aim to temporarily express the Cas9 nuclease in or deliver the protein directly into the target cell. Thus, a putative humoral antibody response may be negligible. 5 However, intracellular protein degradation processes lead to peptide presentation of Cas9 fragments on the cellular surface of gene-edited cells that may be recognized by T cells. While a primary T cell response could be prevented or delayed, a pre-existing memory would have major impact.Here, we show the presence of a ubiquitous memory/effector T cell response directed towards the most popular Cas9 homolog from Streptococcus pyogenes (SpCas9) within healthy human subjects. We have characterized SpCas9-reactive memory/effector T cells (TEFF) within the CD4/CD8 compartments for multi-effector potency and lineage determination. Intriguingly, SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with the magnitude of the respective TEFF response. SpCas9-specific TREG may be harnessed to ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response in vivo. Furthermore, the equilibrium of Cas9-specific TEFF and TREG cells may have greater importance in Streptococcus pyogenes-associated diseases. Our results shed light on the T cell mediated immunity towards the much-praised gene scissor SpCas9 and offer a possible solution to overcome the problem of pre-existing immunity. TextSpCas9 was the first Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated nuclease hijacked to introduce DNA double-strand breaks at specific DNA sequences. 1 Through the ease of target adaption and the remarkable efficacy, it advanced to the most popular tool for re-writing genes in research and potential clinical applications. The major concern for clinical translation of CRISPR/Cas9 technology is the risk for off-target activity causing potentially harmful mutations or chromosomal aberrations. 2,7 High-fidelity Cas9 enzymes were developed to reduce the probability of these events. 8 Furthermore, novel Cas9-based fusion proteins allow base editing or specific epigenetic reprogramming without inducing breaks in the DNA. 9,10 Most approaches are based on the original SpCas9 enzyme that originates in the facultatively

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but are associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL-7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Co-expressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and anti-tumor activity during repeated exposure to tumor cells, without T cell dysfunction or autonomous T cell growth. Furthermore, C7R co-expressing CAR-T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer.

ObjectiveTo assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model.DesignInvestigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13).SettingCharité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union).ParticipantsRecipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9).InterventionsCD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×106 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48.Main outcome measuresThe primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio.ResultsFor all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire.ConclusionsThe application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies.Trial registrationNCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

Chimeric antigen receptors (CAR) are fusion proteins that redirect T cell specificity towards surface molecules expressed on tumour cells independently of the conventional T cell receptor (TCR)-major histocompatibility complex (MHC) interactions. CARs are introduced into T cells through gene transfer 1,2 . The antigen-recognition domain most often consists of a mouse-derived monoclonal antibody as a continuous peptide single-chain variable fragment (scFv) steered through an extracellular spacer domain that provides flexibility. ScFvs engage with their target epitopes and confer activation signals through modular intracellular signalling domains. Currently, CAR T cells are generated ex vivo from peripheral blood-derived T cells, which are typically transduced with replication-deficient vectors that integrate the CAR expression cassette into the T cell genome. These CAR T cells are subsequently expanded to large numbers in culture. After infusion into patients, these cells can recognize and eliminate tumour cells expressing the target antigen.

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(−) escape BL-ALL while preserving their upfront efficacy. Supplementary informationThe online version of this article (https://

Therapies with genetically modified T cells that express chimeric antigen receptors (CARs) specific for CD19 or B cell maturation antigen (BCMA) are approved to treat certain B cell malignancies. However, translating these successes into treatments for patients with solid tumours presents various challenges, including the risk of clinically serious on-target, off-tumour toxicity (OTOT) owing to CAR T cellmediated cytotoxicity against non-malignant tissues expressing the target antigen. Indeed, severe OTOT has been observed in various CAR Review articlewe examine the implications of OTOT on the development of CAR T cell therapies targeting solid tumours, summarize OTOT evidence in preclinical and clinical studies, and discuss advances in CAR T cell engineering that might help to overcome OTOT in the clinic. Risks and mechanisms of OTOTOTOT stems from CAR T cell-mediated recognition and lysis of nonmalignant tissues expressing the target antigen, potentially causing severe adverse events 23,26 . Upon recognition of a target antigen, CAR T cell activation leads to the formation of an immune synapse between the CAR and the target cell 27 , triggering effector functions (Fig. 2). The release of perforin and granzymes 28 is assumed to be a principal mechanism of CAR T cell-mediated cytotoxicity. However, other mechanisms, such as upregulation of T cell-surface molecules to induce target apoptosis (such as FAS ligand) 29 or secretion of cytokines, including IFNγ and/or TNF, may also contribute to tissue destruction [29][30][31] (Fig. 2).To generate CAR T cells that are both safe and effective in patients with solid tumours, target antigen selection is crucial. Optimal antigen candidates, referred to as neoantigens, should be exclusively expressed on malignant cells and not on non-malignant cells. Such antigens could arise from tumour-specific non-synonymous mutations, insertions or deletions that alter the amino acid sequence of cell-surface proteins, aberrant expression of oncofetal antigens, or tumour-specific posttranslational modifications [32][33][34][35][36][37] . However, cell-surface neoantigens are rare, particularly in tumours with a low mutational burden 38 . EGFRvIII, found in 24-67% of glioblastomas, is one of the few identified examples 39,40 . Consequently, the majority of CAR T cell therapy targets for solid tumours are tumour-associated antigens (TAAs) that are also expressed on non-malignant tissues (Fig. 3).