Explore the vast range of titles available.

BackgroundAdoptive transfer of chimeric antigen receptor (CAR)-expressing natural killer (NK) cells has demonstrated success against hematological malignancies. Efficacy against solid tumors has been limited by poor NK cell survival and function in the suppressive tumor microenvironment (TME). To enhance efficacy against solid tumors, stimulatory cytokines have been incorporated into CAR-NK cell therapeutic approaches. However, current cytokine strategies have limitations, including systemic toxicities, exogenous dependencies, and unwanted TME bystander effects. Here, we aimed to overcome these limitations by modifying CAR-NK cells to express a constitutively active interleukin (IL)-7 receptor, termed C7R, capable of providing intrinsic CAR-NK cell activation that does not rely on or produce exogenous signals nor activate bystander cells.MethodsWe examined persistence, antitumor function, and transcriptional profiles of CAR-NK cells coexpressing C7R in a novel tumor immune microenvironment (TiME) co-culture system and against hematologic and solid tumor xenografts in vivo.ResultsPeripheral blood NK cells expressing a CAR directed against the solid tumor antigen GD2 and modified with C7R demonstrated enhanced tumor killing and persistence in vitro compared with CAR-NK cells without cytokine support and similar functions to CAR-NK cells supplemented with recombinant IL-15. C7R.CAR-NK cells exhibited enhanced survival and proliferation within neuroblastoma TiME xenografts in vivo but produced poor long-term tumor control compared with CAR-NK cells supplemented with IL-15. Similar results were seen using C7R-expressing CD19.CAR-NK cells against CD19+leukemia xenografts. Gene expression analysis revealed that chronic signaling via C7R induced a transcriptional signature consistent with intratumor stressed NK cells with blunted effector function. We identified gene candidates associated with chronic cytokine-stressed NK cells that could be targeted to reduce CAR-NK cell stress within the solid TME.ConclusionC7R promoted CAR-NK cell survival in hostile TMEs independent of exogenous signals but resulted in poor antitumor function in vivo. Our data reveals the detrimental role of continuous IL-7 signaling in CAR-NK cells and provides insights into proper application of cytokine signals when attempting to enhance CAR-NK cell antitumor activity.

Natural killer (NK) cells are innate immune effectors capable of broad cytotoxicity via germline-encoded receptors and can have conferred cytotoxic potential via the addition of chimeric antigen receptors. Combined with their reduced risk of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), NK cells are an attractive therapeutic platform. While significant progress has been made in treating hematological malignancies, challenges remain in using NK cell-based therapy to combat solid tumors due to their immunosuppressive tumor microenvironments (TMEs). The development of novel strategies enabling NK cells to resist the deleterious effects of the TME is critical to their therapeutic success against solid tumors. In this review, we discuss strategies that apply various genetic and non-genetic engineering approaches to enhance receptor-mediated NK cell cytotoxicity, improve NK cell resistance to TME effects, and enhance persistence in the TME. The successful design and application of these strategies will ultimately lead to more efficacious NK cell therapies to treat patients with solid tumors. This review outlines the mechanisms by which TME components suppress the anti-tumor activity of endogenous and adoptively transferred NK cells while also describing various approaches whose implementation in NK cells may lead to a more robust therapeutic platform against solid tumors.

BackgroundNatural killer cells (NKs) expressing chimeric antigen receptors (CAR-NKs) were successful in hematological malignancies.1 However, solid tumors resist CAR-NKs via a tumor microenvironment (TME) that includes myeloid derived suppressor cells (MDSCs) and M2 macrophages (M2s).2 We demonstrated that ex vivo manufacture of therapeutic CAR-NKs significantly upregulated T cell immunoreceptor with Ig and ITIM domains (TIGIT), an inhibitory NK receptor. Analysis of pediatric neuroblastoma and sarcoma patient tumors confirmed high expression of TIGIT ligands on tumor cells and intra-tumoral MDSCs and M2s. Our main objective was to determine influence of TIGIT on CAR-NK function in the TME. Current TIGIT-targeting approaches using antibodies are handicapped by poor bioavailability and transient binding in the TME. We hypothesized that genetic deletion of TIGIT on CAR-NKs will lead to a more profound and durable anti-tumor response within the TME.3MethodsTIGIT knockout (KO) CAR-NKs expressing a GD2.4-1BB.zeta CAR were generated by concurrent CRISPR/cas9 and retroviral transduction of expanded primary human NK cells. Degranulation (CD107a) and IFN-γ by TIGITKO.GD2.CAR-NK were assessed in short-term TME co-cultures containing LAN-1 neuroblastoma and human MDSCs. A novel long-term TME co-culture wherein human monocytes and CHLA255 neuroblastoma pre-established a suppressive TME for 72 hours prior to addition of TIGITKO GD2.CAR-NK was used to assess tumor growth and CAR-NK proliferation over 96 hours using Incucyte. We assessed phosphorylated mTOR (pMTOR) in CAR-NKs by intracellular flow cytometry.ResultsCRISPR/Cas9 and retroviral transduction generated stable TIGITKO.CAR-NKs (>90% TIGIT deletion; 60% GD2.CAR expression). TIGITKO enhanced GD2.CAR-NK cytokine secretion but not degranulation in short-term TME co-cultures. In long-term tumor co-cultures, TIGITKO.GD2.CAR-NKs eliminated more tumor vs control NKs (<10% viable tumor vs 35% viable tumor, p<0.01; n=4 donors). In TME co-cultures, TIGITKO.GD2.CAR-NK rapidly proliferated and controlled tumor compared to TIGITwt.CAR-NKs (37% viable tumor vs 57% viable tumor, p<0.05; n=4 donors). TIGITKO did not increase CAR-NK degranulation or Fas ligand expression, nor did it alter CAR-NK surface expression of DNAM-1, NKG2D, NKG2A, TIM-3, PD-1 or LAG-3. While TIGITwt.CAR-NKs massively downregulated pMTOR within the TME, TIGITKO.CAR-NKs maintained pMTOR expression. Ongoing studies using a novel in vivo TME xenograft model of neuroblastoma will determine benefit of TIGITKO vs. TIGIT antibody therapy on CAR-NK activity.ConclusionsWe defined a role for TIGIT in inhibition of CAR-NK function and suggest TIGIT deletion as a novel NK therapeutic platform to evade immune suppression in the TME. This highlights the potential of gene-edited CAR-NKs to improve clinical outcomes in patients with solid tumors.ReferencesLiu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, Nassif Kerbauy L, Overman B, Thall P, Kaplan M, Nandivada V, Kaur I, Nunez Cortes A, Cao K, Daher M, Hosing C, Cohen EN, Kebriaei P, Mehta R, Neelapu S, Nieto Y, Wang M, Wierda W, Keating M, Champlin R, Shpall EJ, Rezvani K. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med. 2020;382: 545–553.Beavis PA, Slaney CY, Kershaw MH, Gyorki D, Neeson PJ, Darcy PK. Reprogramming the tumor microenvironment to enhance adoptive cellular therapy. Semin. Immunol. 2016;28: 64–72.Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol. 2009. 157: 220–233.

Detailed field mapping of glacial and paraglacial landforms and optical dating from these landforms are used to reconstruct the early Holocene glaciation in the semi-arid region of Miyar basin, Lahaul Himalaya. The study identifies three stages of glaciation, of decreasing magnitude and termed, from oldest to youngest, the Miyar stage (MR-I), Khanjar stage (KH-II), and Menthosa advance (M-III). The oldest glacial stage (MR-I) has been established on the basis of detailed geomorphological evidence such as U-shaped valley morphology, trimlines, and truncated spurs. It is speculated to be older than the global Last Glacial Maximum (gLGM) based on the magnitude of ΔELA (Equilibrium-Line Altitude, 606m). No evidence of glacier expansion recorded from the basin correlates with the period of the gLGM. The second stage (KH-II) is well represented by extensive depositional features such as lateral and terminal moraines, drumlins, and lacustrine fills that have been constrained within 10 ± 1 to 6.6 ± 1.0 ka (Optically stimulated luminescence—OSL—ages), dating it to the early Holocene advance following the Younger Dryas cooling event. Exceptionally young glacial records of expansion are limited within a few hundred meters of the present termini of tributary glaciers and correlates with the 18th-century cooling event. Records of this glacial advance, termed the Menthosa advance, are clearly noticed in some tributary valleys.

Macrocyclic complexes of co(III) of the type, CoCy1-2B’X where Cy1 represents themacrocyclic ligand 1,8-dichloro-2,7,9,14-tetraoxa-1,1,8,8-tetraisopropoxy- 4,5,11,12-tetramethyl-1,3,6,10,13-tetraaza, cyclotetradeca-3,5,10,12- tetrene[boimacylene(14)] and cy2-represents the macrocyclic ligand 1,8-dibora- 2,7,9,14-tetraoxa -1,1,8,8-tetraisopropoxy 4,5,11,12-tetraphenyl-1,3,6,10,13-Tetraazacyclotetradeca-3,5,10,12-tetrene [boiphacyclene(14) ]; X represents Halide ions and B’represents nitrogen- donor bases such as pyridine or 3-or 4- methyl pyridines, have been prepared 1-2 and characterized on the basis of elemental analysis, spectral studies and electrochemical studies. All the complexes are found to posses octahedral symmetry around the Co(III)ion.