Preclinical NK Cell Platform for CAR Directed Therapies: Functional and Phenotypic Comparison Using a Rechallenge Cytotoxicity Assay

Introduction: CAR T-cell therapy has revolutionized the treatment of patients with relapsed/refractory (R/R) acute leukemia, NHL, and multiple myeloma. However, there are still areas of improvement in their clinical activity, source of the effector cells, prevention, and management of adverse events that require particular attention. Because of those reasons, NK cells appear as a viable effector cell alternative that can help address these challenges. NK cells offer a profile of activation, expansion, persistence, and cytotoxicity that is different from T cells and, when modified to bear CAR constructs, may provide significant advantages. However, the preclinical development of NK-CARs is challenging mainly because of the difficulty of generating large quantities of cells for testing and well-established pathways for CAR optimization before in vivo evaluation. Therefore, we developed a CAR optimization platform using the NK-92 cell line. NK-92 cells conserve their cytotoxic ability and can be easily expanded in vitro and used for functional and phenotypical evaluations of novel CAR-NK constructs. Here we present a rechallenge cytotoxic assay that mimics repetitive in vivo effector interactions with the target cells and its use for optimization, comparison, and development of NK-based cellular therapies. Methods: We generated lentivirus transduced CD19 CARs (FMC63-41BB-z) using T cells from healthy donors and NK-92 cells for comparison.T cells were expanded for 12 days, and a 41.9% CAR+ expression was achieved (CART19). Transduced NK-92 cells were sorted by FACS to obtain a population of 98.3 % CAR+ cells (CARNK19) and subsequently expanded for 12 days. JeKo-1 cells were used as CD19+ targets and BxPC3 cells as CD19 neg control (both cell types were GFP-Luc-PuroR). We developed a Luciferase-based rechallenge cytotoxicity assay. For this, we diluted the effector to target (E/T) ratio to obtain a logarithmic trendline of the cells' cytotoxicity. E/T ratio to get viability of 50% (IC50) measured at 4h (for CARNK19) and 24h (for CART19) was used as a proxy of the product's potency. Both CAR Immune Effector Cells (IECs) were co-cultured with their targets at an E/T ratio to obtain 70% cytotoxicity. After 24 hours with the target, we estimated the remaining IEC amount in the culture using GFP exclusion in flow analysis (IEC cells/mL = total cells/mL x GFP neg%). We repeated the plating of E/T ratio dilutions to perform daily IC50 curves using this rechallenge strategy for a total of 5 days. CAR and PD1 expression were measured on Day 0 and Day 5 by flow cytometry. Results: CART19 showed a higher IC50 than CARNK19 at baseline, 1.7 vs. 0.19 (Figure 1A). The IC50 trend of both IECs over time showed an uptrend that suggests progressive functional exhaustion (Figure 1B). At 5 days of rechallenge, it was 29 times higher in T cells than in NK-92 (12.07 vs. 0.42) and with a slope 265 times higher (10.6 vs. 0.04). Furthermore, we observed that when comparing the levels of CAR expression on Day 0 vs. Day 5, CART19 showed a decrease in CAR expression that was not present in CARNK19 (41.9 to 10.9% vs. 98.3 to 95.5%) (Figure 1C). In addition, there was a higher increase in PD1 expression in CART19 cells than CARNK19 cells from Day 0 to Day 5 of the in vitro rechallenge (9.9 to 46.8% vs. 0.88 to 8.88%) (Figure 1D). Conclusion: Our data shows the use of NK-92 cells as a tool for optimization and preclinical development of NK cell-based cellular therapies. We demonstrated that it is feasible to set up repetitive cytotoxic challenges that mimic closer in vivo E/T engagement. Moreover, using the cytotoxic IC50 calculated with this platform, we show increased cytotoxicity, less functional exhaustion, and less expression of PD1 in CARNK19 than in its T cell counterpart. Overall, the NK-92 rechallenge cytotoxicity assay platform constitutes a helpful tool for research, development, and optimization of cellular therapies based on NK cell effector function. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Accelerated Progression of Disseminated Coccidioidomycosis Following SARS-CoV-2 Infection: A Case Report

ABSTRACT We describe a patient with subclinical coccidioidomycosis who experienced rapid disease dissemination shortly after SARS-CoV-2 infection, suggesting host immune response dysregulation to coccidioidomycosis by SARS-CoV-2. We hypothesize that disrupted cell-mediated signaling may result after SARS-CoV-2 infection leading to functional exhaustion and CD8+ T-cell senescence with impairment in host cellular response to Coccidioides infection.

Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

Checkpoint blockade can reverse CD8+ T-cell functional exhaustion, and TCF-1 is essential for this process. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence in checkpoint blockade non-responsive tumors remains a challenge. We demonstrate that targeting Cbx3/HP1γ causes augmented transcription initiation, chromatin remodeling at Lef1 and Il21r leading to increased transcriptional activity at these loci. Mechanistic studies show LEF-1 and IL-21R are required for Cbx3/HP1γ-deficient CD8+ effector T cells to persist resulting in improved control of ovarian cancer, melanoma and neuroblastoma in pre-clinical models. Cbx3/HP1γ-deficient CD8+ T cells enhanced persistence in the TME facilitates remodeling of the chemokine/receptor landscape that ensures their optimal tumor invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by checkpoint blockade, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for resistant, non-responsive solid tumors.

Functional Exhaustion of Type I and II Interferons Production in Severe COVID-19 Patients

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged in Wuhan in December 2019 and has since spread across the world. Even though the majority of patients remain completely asymptomatic, some develop severe systemic complications. In this prospective study we compared the immunological profile of 101 COVID-19 patients with either mild, moderate or severe form of the disease according to the WHO classification, as well as of 50 healthy subjects, in order to identify functional immune factors independently associated with severe forms of COVID-19. Plasma cytokine levels, and cytokine levels upon in vitro non-specific stimulation of innate and adaptive immune cells, were measured at several time points during the course of the disease. As described previously, inflammatory cytokines IL1β, IL6, IL8, and TNFα associated with cytokine storm were significantly increased in the plasma of moderate and severe COVID-19 patients (p < 0.0001 for all cytokines). During follow-up, plasma IL6 levels decreased between the moment of admission to the hospital and at the last observation carried forward for patients with favorable outcome (p = 0.02148). After in vitro stimulation of immune cells from COVID-19 patients, reduced levels of both type I and type II interferons (IFNs) upon in vitro stimulation were correlated with increased disease severity [type I IFN (IFNα): p > 0.0001 mild vs. moderate and severe; type II IFN (IFNγ): p = 0.0002 mild vs. moderate and p < 0.0001 mild vs. severe] suggesting a functional exhaustion of IFNs production. Stimulated IFNα levels lower than 2.1 pg/ml and IFNγ levels lower than 15 IU/mL at admission to the hospital were associated with more complications during hospitalization (p = 0.0098 and p =0.0002, respectively). A low IFNγ level was also confirmed by multivariable analysis [p = 0.0349 OR = 0.98 (0.962; 0.999)] as an independent factor of complications. In vitro treatment with type IFNα restored type IFNγ secretion in COVID-19 patients while the secretion of pro-inflammatory cytokines IL6 and IL1β remained stable or decreased, respectively. These results (a) demonstrate a functional exhaustion of both innate and adaptive immune response in severe forms of COVID-19; (b) identify IFNα and IFNγ as new potential biomarkers of severity; and (c) highlight the importance of targeting IFNs when considering COVID-19 treatment in order to re-establish a normal balance between inflammatory and Th1 effector cytokines.