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.

Immunoglobulin E (IgE) is central in allergy pathology as it binds high-affinity receptors on mast cells, inducing degranulation upon allergen crosslinking. However, IgE has the lowest serum concentration and shortest half-life of all Ig’s, and IgE+ B cells are extremely rare. This work aims to uncover characteristics of IgE+B cells that could be used in the design of new therapeutic strategies for allergic diseases. IgE+ cells were identified in Atopic Dermatitis (AD) patients, and were also generated in vitro by stimulating with aCD40 and IL-4. Naïve, IgG+, IgA+ and IgE+ cells from AD donors were phenotyped and sorted by flow cytometry, and their transcriptomes were determined by RNAseq. Inter-sample similarity was determined using multidimensional scaling (MDS). Gene ontology (GO) of differentially expressed genes (DEG) identified molecular pathways enriched in IgE+ cells. IgE+ cells were CD38hiCD27hi, indicating a high content of plasma cells in IgE+ B cells. IgE+ cells expressed high levels of proliferation marker Ki67. MDS comparison of datasets of naïve, IgG+, IgA+ and IgE+ cells showed that IgE cells were distant from both IgG and IgA cells. GO analysis of IgE DEG demonstrated that IgE cells expressed genes associated with N-glycosylation, the IRE1-XBP1 endoplasmic reticulum unfolded protein response, and mitochondrial oxidative phosphorylation, synonymous with plasma cells. Our data indicates that IgE+ cells in AD patients are predominantly plasmablasts and plasma cells, and not memory B cells. Ongoing studies aim to characterize human IgE cell diversity using single cell analysis, and to identify non-IgE precursor memory cells that give rise to IgE+ cells upon allergen activation.

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Background Natural 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 antitumor response within the TME. 3 Methods TIGIT 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-g by TIGIT KO . GD2.

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Methods We generated MUC18-cytotoxic and csCARs with 4-1BB, OX40, 2B4, and DNAM-1 endodomains and confirmed specificity and functionality using MUC18 overexpressing and knockout targets and a long-term TME co-culture comprised of alveolar rhabdomyosarcoma, Rh4, and inhibitory macrophages (M2s). Safety of MUC18-csCARs was tested against the MUC18+ liver sinusoidal endothelial cell (LSEC) line. Anti-tumor activity of dual-targeted NK cells compared to unmodified and singly-modified NK cells was assessed in vivo using a novel TME xenograft model with Rh4 and MDSCs. Results MUC18 cytotoxic CAR-NK cells killed MUC+ high targets, while exhibiting low killing against an Rh4-MUC18 KO cell line, confirming CAR specificity and function. MUC18-OX40csCAR NK cells expanded without additional killing in the TME compared to NK cells with other co-stimulatory endodomains. MUC18-OX40csCAR NK cells did not exhibit killing of LSECs. Dual-targeted NK cells demonstrated enhanced tumor control in TME co-cultures (2.4-fold change in tumor vs. 4.6 by unmodified NK, 10.6 by NKG2D.z, and 6.8 by cs.MUC18) compared to either singly-modified NK population (figure 1). Dual-targeted NK cells demonstrated superior tumor control in the in vivo TME xenograft model compared to controls (p=0.007 versus NKG2D.z) and prolonged survival (p= <0.0001) (figure 2). Conclusions Dual-targeted NK cells demonstrate enhanced anti-tumor activity without toxicity against normal tissue. Use of co-stimulation-only CARs in NK cells may allow exploitation of previously non-targetable sarcoma antigens. Abstract 394 Figure 1 The dual-targeted CAR NK cells were compared to unmodified NK cells, NKG2D.? CAR NK cells, and csMUC18 CAR NK cells in a TME co-culture system. A) Rh4 fold expansion in the tumor alone conditions and B) in the TME conditions was determined for each time point. C) Fold change in NK cells in the tumor alone conditions. D) Fold change in NK cells in the TME conditions. Abstract 394 Figure 2 (A) Mice (n=5 per treatment group) received two NK cells doses at 31 and 38 days post tumor inoculation and three times per week IL-2 and IL-15 to promote NK cell survival. (B) Survival probability of each treatment cohort.

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