Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

Localized in vivo gene editing of murine cancer-associated fibroblasts.

Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of Osmr, Tgfbr2, and Il1r1 in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an in vivo knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated in vivo gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond.

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2 + influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca + + influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2 + flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70. Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

Structural surfaceomics reveals an AML-specific conformation of integrin ß2 as a CAR T cellular therapy target.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

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.

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes.

CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.

Novel extragenic genomic safe harbors for precise therapeutic T-cell engineering.

Cell therapies that rely on engineered immune cells can be enhanced by achieving uniform and controlled transgene expression in order to maximize T-cell function and achieve predictable patient responses. Although they are effective, current genetic engineering strategies that use γ-retroviral, lentiviral, and transposon-based vectors to integrate transgenes, unavoidably produce variegated transgene expression in addition to posing a risk of insertional mutagenesis. In the setting of chimeric antigen receptor (CAR) therapy, inconsistent and random CAR expression may result in tonic signaling, T-cell exhaustion, and variable T-cell persistence. Here, we report and validate an algorithm for the identification of extragenic genomic safe harbors (GSH) that can be efficiently targeted for DNA integration and can support sustained and predictable CAR expression in human peripheral blood T cells. The algorithm is based on 7 criteria established to minimize genotoxicity by directing transgene integration away from functionally important genomic elements, maximize efficient CRISPR/Cas9-mediated targeting, and avert transgene silencing over time. T cells engineered to express a CD19 CAR at GSH6, which meets all 7 criteria, are curative at low cell dose in a mouse model of acute lymphoblastic leukemia, matching the potency of CAR T cells engineered at the TRAC locus and effectively resisting tumor rechallenge 100 days after their infusion. The identification of functional extragenic GSHs thus expands the human genome available for therapeutic precision engineering.

An evolved AAV variant enables efficient genetic engineering of murine T cells.

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.

Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

NUDT21 limits CD19 levels through alternative mRNA polyadenylation in B cell acute lymphoblastic leukemia.

B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.

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.

Generation of T-cell-receptor-negative CD8αß-positive CAR T cells from T-cell-derived induced pluripotent stem cells.

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of 'off-the-shelf' CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR- CD8αß+ CAR T cells that perform similarly to CD8αß+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αß+ T cells for a broad range of immunotherapies.

The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma.

HLA-independent T cell receptors for targeting tumors with low antigen density.

Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.

The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28.

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG4) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG4-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.

CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape.

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4-9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.

Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency.

Chimeric antigen receptors (CARs) are synthetic receptors that target and reprogram T cells to acquire augmented antitumor properties1. CD19-specific CARs that comprise CD28 and CD3ζ signaling motifs2 have induced remarkable responses in patients with refractory leukemia3-5 and lymphoma6 and were recently approved by the US Food and Drug Administration7. These CARs program highly performing effector functions that mediate potent tumor elimination4,8 despite the limited persistence they confer on T cells3-6,8. Extending their functional persistence without compromising their potency should improve current CAR therapies. Strong T cell activation drives exhaustion9,10, which may be accentuated by the redundancy of CD28 and CD3ζ signaling11,12 as well as the spatiotemporal constraints imparted by the structure of second-generation CARs2. Thus, we hypothesized that calibrating the activation potential of CD28-based CARs would differentially reprogram T cell function and differentiation. Here, we show that CARs encoding a single immunoreceptor tyrosine-based activation motif direct T cells to different fates by balancing effector and memory programs, thereby yielding CAR designs with enhanced therapeutic profiles.

Low-Dose Radiation Conditioning Enables CAR T Cells to Mitigate Antigen Escape.

CD19 chimeric antigen receptors (CARs) have demonstrated great efficacy against a range of B cell malignancies. However, antigen escape and, more generally, heterogeneous antigen expression pose a challenge to applying CAR therapy to a wide range of cancers. We find that low-dose radiation sensitizes tumor cells to immune rejection by locally activated CAR T cells. In a model of pancreatic adenocarcinoma heterogeneously expressing sialyl Lewis-A (sLeA), we show that not only sLeA+ but also sLeA- tumor cells exposed to low-dose radiation become susceptible to CAR therapy, reducing antigen-negative tumor relapse. RNA sequencing analysis of low-dose radiation-exposed tumors reveals the transcriptional signature of cells highly sensitive to TRAIL-mediated death. We find that sLeA-targeted CAR T cells produce TRAIL upon engaging sLeA+ tumor cells, and eliminate sLeA- tumor cells previously exposed to systemic or local low-dose radiation in a TRAIL-dependent manner. These findings enhance the prospects for successfully applying CAR therapy to heterogeneous solid tumors. Local radiation is integral to many tumors' standard of care and can be easily implemented as a CAR conditioning regimen.

Antibody with Infinite Affinity for In Vivo Tracking of Genetically Engineered Lymphocytes.

There remains an urgent need for the noninvasive tracking of transfused chimeric antigen receptor (CAR) T cells to determine their biodistribution, viability, expansion, and antitumor functionality. DOTA antibody reporter 1 (DAbR1) comprises a single-chain fragment of the antilanthanoid-DOTA antibody 2D12.5/G54C fused to the human CD4-transmembrane domain and binds irreversibly to lanthanoid (S)-2-(4-acrylamidobenzyl)-DOTA (AABD). The aim of this study was to investigate whether DAbR1 can be expressed on lymphocytes and used as a reporter gene as well as a suicide gene for therapy of immune-related adverse effects. Methods: DAbR1 was subcloned together with green fluorescent protein into an SFG-retroviral vector and used to transduce CD3/CD28-activated primary human T cells and second-generation 1928z (CAR) T cells. Cell surface expression of DAbR1 was confirmed by cell uptake studies with radiolabeled AABD. In addition, the feasibility of imaging of DAbR1-positive T cells in vivo after intravenous injection of 86Y/177Lu-AABD was studied and radiation doses determined. Results: A panel of DAbR1-expressing T cells and CAR T cells exhibited greater than 8-fold increased uptake of 86Y-AABD in vitro when compared with nontransduced cells. Imaging studies showed 86Y-AABD was retained by DAbR1-positive T cells while it continuously cleared from normal tissues, allowing for in vivo tracking of intravenously administered CAR T cells. Normal-organ dose estimates were favorable for repeated PET/CT studies. Selective T cell ablation in vivo with 177Lu-AABD seems feasible for clustered T-cell populations. Conclusion: We have demonstrated for the first time that T cells can be modified with DAbR1, enabling their in vivo tracking via PET and SPECT. The favorable biodistribution and high image contrast observed warrant further studies of this new reporter gene.