Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Cost-effectiveness analyses of targeted therapy and immunotherapy for advanced non-small cell lung cancer in the United States: a systematic review.

Introduction: Mutation-targeting and immuno-oncology drugs are revolutionizing the treatment of advanced non-small cell lung cancer (NSCLC). Cost-effectiveness analyses (CEA) of these drugs have been conducted using various analytical methods and cost-effectiveness thresholds. This systematic review provides a comprehensive summary of the available evidence.Area covered: PubMed, Embase, and Cochrane Library were used to select for CEA of targeted therapies for NSCLC in the United States published between 2008 and 2020. Among the 28 included studies, a majority were published from 2017 to 2020 (n = 18) and more than half targeted non-squamous NSCLC (n = 15). The most frequently evaluated therapy was pembrolizumab (n = 11), followed by bevacizumab (n = 8) and erlotinib (n = 4). After 2009, all included studies applied $100,000 or more thresholds. Thresholds of studies supported by industry (median = $150,000) were more distributed than those of studies supported by nonprofits (median = $100,000).Expert commentary: Medications of interest have changed and are individualized to particular mutations. The cost-effectiveness thresholds varied among sponsors but generally trended to increase over time. This review provides an overview of the available cost-effectiveness findings for stakeholders and contributes to evidence-based practice.

A semi-interpenetrating network of polyacrylamide and recombinant basement membrane allows pluripotent cell culture in a soft, ligand-rich microenvironment.

The physical properties of the extracellular matrix play an essential role in guiding stem cell differentiation and tissue morphogenesis both in vivo and in vitro. Existing work to investigate the role of matrix mechanics in directing stem cell proliferation, self-renewal, and differentiation has been limited by the poor attachment and survival of human pluripotent cells cultured on soft matrices (Young's modulus E ≲ 1000 Pa). To address this limitation we developed a protocol for generating semi-interpenetrating networks of polyacrylamide and recombinant basement membrane. Using these materials, we found that human embryonic stem cells (hESCs) remained proliferative and pluripotent even when grown in small colonies and on surfaces ranging in stiffness from 150 to 12000 Pa, spanning the range of tissue stiffnesses likely to be encountered in the embryo. Considerable recent attention has focused on the role of the transcriptional coactivator and Hippo effector YAP in regulating differentiation and cell proliferation both in the early embryo and in vitro. We found that while YAP localized to the nucleus on substrates of E ≳ 1000 Pa, its localization was heterogeneous on substrates of moduli ≲ 450 Pa, with predominantly nuclear localization at the colony periphery and mixed cytoplasmic and nuclear localization for cells in the colony interior, a pattern reminiscent of YAP subcellular localization in the inner cell mass (ICM) of the early embryo. In addition, hESC colony dynamics were highly responsive to substrate stiffness, with cells assembling into monolayers, multilayer structures, and transient, hollow rosettes in response to decreasing substrate stiffnesses in the range of 12000 to 150 Pa. We suggest that soft, ligand-rich substrates such as are described here provide a promising means of recapitulating aspects of early mammalian development that are otherwise inaccessible, and more broadly may be useful in the derivation of complex tissues from pluripotent cells in an in vitro setting.

Sphingosine 1-phosphate receptor 1 regulates the directional migration of lymphatic endothelial cells in response to fluid shear stress.

The endothelial cells that line blood and lymphatic vessels undergo complex, collective migration and rearrangement processes during embryonic development, and are known to be exquisitely responsive to fluid flow. At present, the molecular mechanisms by which endothelial cells sense fluid flow remain incompletely understood. Here, we report that both the G-protein-coupled receptor sphingosine 1-phosphate receptor 1 (S1PR1) and its ligand sphingosine 1-phosphate (S1P) are required for collective upstream migration of human lymphatic microvascular endothelial cells in an in vitro setting. These findings are consistent with a model in which signalling via S1P and S1PR1 are integral components in the response of lymphatic endothelial cells to the stimulus provided by fluid flow.

Multiplexed Fluid Flow Device to Study Cellular Response to Tunable Shear Stress Gradients.

Endothelial cells (ECs) line the interior of blood and lymphatic vessels and experience spatially varying wall shear stress (WSS) as an intrinsic part of their physiological function. How ECs, and mammalian cells generally, sense spatially varying WSS remains poorly understood, due in part to a lack of convenient tools for exposing cells to spatially varying flow patterns. We built a multiplexed device, termed a 6-well impinging flow chamber, that imparts controlled WSS gradients to a six-well tissue culture plate. Using this device, we investigated the migratory response of lymphatic microvascular ECs, umbilical vein ECs, primary fibroblasts, and epithelial cells to WSS gradients on hours to days timescales. We observed that lymphatic microvascular ECs migrate upstream, against the direction of flow, a response that was unique among all the cells types investigated here. Time-lapse, live cell imaging revealed that the microtubule organizing center relocated to the upstream side of the nucleus in response to the applied WSS gradient. To further demonstrate the utility of our device, we screened for the involvement of canonical signaling pathways in mediating this upstream migratory response. These data highlight the importance of WSS magnitude and WSS spatial gradients in dictating the cellular response to fluid flow.