Peripheral NK Research Articles

A tetraspecific engager armed with a non-alpha IL-2 variant harnesses natural killer cells against B cell non-Hodgkin lymphoma.

NK cells offer a promising alternative to T cell therapies in cancer. We evaluated IPH6501, a clinical-stage, tetraspecific NK cell engager (NKCE) armed with a non-alpha IL-2 variant (IL-2v), which targets CD20 and was developed for treating B cell non-Hodgkin lymphoma (B-NHL). CD20-NKCE-IL2v boosts NK cell proliferation and cytotoxicity, showing activity against a range of B-NHL cell lines, including those with low CD20 density. Whereas it presented reduced toxicities compared with those commonly associated with T cell therapies, CD20-NKCE-IL2v showed greater killing efficacy over a T cell engager targeting CD20 in in vitro preclinical models. CD20-NKCE-IL2v also increased the cell surface expression of NK cell-activating receptors, leading to activity against CD20-negative tumor cells. In vivo studies in nonhuman primates and tumor mouse models further validated its efficacy and revealed that CD20-NKCE-IL2v induces peripheral NK cell homing at the tumor site. CD20-NKCE-IL2v emerges as a potential alternative in the treatment landscape of B-NHL.

Three-Dimensional Model Analysis Revealed Differential Cytotoxic Effects of the NK-92 Cell Line and Primary NK Cells on Breast and Ovarian Carcinoma Cell Lines Mediated by Variations in Receptor-Ligand Interactions and Soluble Factor Profiles.

Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

Peripheral NK cell count predicts response and prognosis in breast cancer patients underwent neoadjuvant chemotherapy.

The count of lymphocyte subsets in blood can reflect the immune status of the body which is closely related to the tumor immune microenvironment and the efficacy of NAT. This study aims to explore the relationship between peripheral blood lymphocyte subsets and the efficacy and prognosis of NAT in breast cancer. We retrospectively analyzed clinicopathological information and peripheral blood lymphocyte subpopulation counts of patients receiving NAT from January 2015 to November 2021 at Sun Yat-sen University Cancer Center. Kaplan-Meier curves were used to estimate the survival probability. The independent predictors of NAT response and survival prognosis were respectively analyzed by multivariate logistic regression and Cox regression, and nomograms were constructed accordingly. The prediction efficiency of three nomograms was validated separately in the training cohort and the testing cohort. 230 patients were included in the study, consisting of 161 in the training cohort and 69 in the testing cohort. After a median follow-up of 1238 days, patients with higher NK cell value showed higher pCR rates and higher OS and RFS after NAT (all P < 0.001). Multivariate analyses suggested NK cell count was an independent predictor of NAT response, OS and RFS. We then built nomograms accordingly and validated the prediction performance in the testing cohort (C index for NAT response: 0.786; for OS: 0.877, for RFS: 0.794). Peripheral blood NK cell count is a potential predictive marker for BC patients receiving NAT. Nomograms based on it might help predict NAT response and prognosis in BC.

Natural Killer Cell Dysfunction In Human Bladder Cancer Is Caused By Tissue-Specific Suppression of SLAMF6 Signaling.

NK cells are innate lymphocytes critical for surveillance of viruses and tumors, however the mechanisms underlying NK cell dysfunction in cancer are incompletely understood. We assessed the effector function of NK cells from bladder cancer patients and found severe dysfunction in NK cells derived from tumors versus peripheral blood. While both peripheral and tumor-infiltrating NK cells exhibited conserved patterns of inhibitory receptor over-expression, this did not explain the observed defects in NK surveillance in bladder tumors. Rather, TME-specific TGF-β and metabolic perturbations such as hypoxia directly suppressed NK cell function. Specifically, an oxygen-dependent reduction in signaling through SLAMF6 was mechanistically responsible for poor NK cell function, as tumor-infiltrating NK cells cultured ex vivo under normoxic conditions exhibited complete restoration of function, while deletion of SLAMF6 abrogated NK cell cytolytic function even under normoxic conditions. Collectively, this work highlights the role of tissue-specific factors in dictating NK cell function, and implicates SLAMF6 signaling as a rational target for immuno-modulation to improve NK cell function in bladder cancer.

Changes in peripheral NK cells during metastatic triple-negative breast cancer progression

Changes in peripheral NK cells during metastatic triple-negative breast cancer progression

Uncovering the Expression Pattern of the Costimulatory Receptors ICOS, 4-1BB, and OX-40 in Exhausted Peripheral and Tumor-Infiltrating Natural Killer Cells from Patients with Cervical Cancer.

Cervical cancer (CC) poses a significant health burden, particularly in low- and middle-income countries. NK cells play a crucial role against CC; however, they can become exhausted and lose their cytotoxic capacity. This work explores the expression of costimulatory receptors (ICOS, 4-1BB, OX-40) in exhausted NK cells from CC patients. Peripheral blood and tumor biopsies were collected, and flow cytometry was used to evaluate the expression of costimulatory receptors in exhausted NK cells. There is an increase of peripheral exhausted NK cells (PD-1+TIGIT+) in CC patients; this subpopulation has a selectively increased expression of the costimulatory receptors ICOS and 4-1BB. An exhausted population is also highly increased in tumor-infiltrating NK cells, and it shows a dramatically increased expression of the costimulatory receptors ICOS (>15×) and 4-1BB (>10×) compared to peripheral NK cells. The exhausted cells, both in the periphery and in the tumor infiltrating lymphocytes (TILs), are also more likely than non-exhausted NK cell populations (PD-1-TIGIT-) to express these costimulatory receptors; increases ranging from 2.0× ICOS, 2.4× 4-1BB, and 2.6× OX-40 in CD56dim PBMCs to 1.5× ICOS, 5× 4-1BB, and 10× OX-40 in TILs were found. Our study demonstrates for the first time the increased expression of the costimulatory receptors ICOS, 4-1BB, and OX-40 in peripheral CD56dim, CD56bright, and tumor-infiltrating NK cells in CC. Targeting these receptors for stimulation could reverse exhaustion and be a promising immunotherapy strategy.

Abstract 1340: Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™

Abstract Natural Killer (NK) cells are highly responsive cytotoxic immune cells of the innate immune system with well characterized anti-tumor properties. Their ability to directly kill malignant cells and elicit an adaptive immune response makes them a promising candidate for a precision guided immunotherapy in oncology. Bicycle® peptides are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle® platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA™). The NK-TICA™ consists of chemically coupled Bicycle® peptides that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell function. We demonstrate potent, selective binding of our Bicycle® peptides to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle® NK-TICA™ as a multifunctional immune cell engager will promote the modulation and anti-tumor activity of peripheral and intra-tumoral NK cells to solid tumors. Citation Format: Fay Dufort, Tucker Ezell, Alexandra Rezvaya, Kathleen Ho, Christopher Leitheiser, Philip Brandish, Kevin McDonnell, Nicholas Keen. Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1340.

Immunological parameters of maternal peripheral blood as predictors of future pregnancy outcomes in patients with unexplained recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (URPL), affecting approximately 1%-5% of women, exhibits a strong association with various maternal factors, particularly immune disorders. However, accurately predicting pregnancy outcomes based on the complex interactions and synergistic effects of various immune parameters without an automated algorithm remains challenging. In this historical cohort study, we analyzed the medical records of URPL patients treated at Xiangya Hospital, Changsha, China, between January 2020 and October 2022. The primary outcomes included clinical pregnancy and miscarriage. Predictors included complement, autoantibodies, peripheral lymphocytes, immunoglobulins, thromboelastography findings, and serum lipids. Least absolute shrinkage and selection operator (LASSO) analysis and logistic regression analysis was performed for model development. The model's performance, discriminatory, and clinical applicability were assessed using area under the curve (AUC), calibration curve, and decision curve analysis, respectively. Additionally, models were visualized by constructing dynamic and static nomograms. In total, 502 patients with URPL were enrolled, of whom 291 (58%) achieved clinical pregnancy and 211 (42%) experienced miscarriage. Notable differences in complement, peripheral lymphocytes, and serum lipids were observed between the two outcome groups. Moreover, URPL patients with elevated peripheral NK cells (absolute counts and proportion), decreased complement levels, and dyslipidemia demonstrated a significantly increased risk of miscarriage. Four models were developed in this study, of which Model 2 demonstrated superior performance with only seven predictors, achieving an AUC of 0.96 (95% CI: 0.93-0.99) and an accuracy of 0.92. A web-based platform was established to visually present model 2 and to facilitate its utilization by clinicians in outpatient settings (available from: https://yingrongli.shinyapps.io/liyingrong/). Our findings suggest that the implementation of such prediction models could serve as valuable tools for providing comprehensive information and facilitating clinicians in their decision-making processes.

Tissue damage from chronic liver injury inhibits peripheral NK cell abundance and proinflammatory function.

One of the difficulties in the treatment of hepatocellular carcinoma is that it is impossible to eliminate the inhibitory effect of the tumor microenvironment on immune response. Therefore, it is particularly important to understand the formation process of the tumor microenvironment. Chronic inflammation is the core factor of cancer occurrence and the leading stage of inflammation-cancer transformation, and the natural killer cell subsets play an important role in it. Our study confirmed that in the stage of chronic liver injury, the local immunosuppressive microenvironment of the liver (i.e. the damaged microenvironment) has been formed, but this inhibitory effect is only for peripheral natural killer cells and has no effect on tissue-resident natural killer subsets. The markers of damage microenvironment are the same as those of tumor microenvironment.

Androgen receptor blockade to activate NK cells and to upregulate the surface expression of HLA-E in prostate cancer cell lines.

190 Background: The blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but most patients progress to castration-resistant prostate cancer (mCRPC). AR signaling modulates CD8+ T cytotoxic function, revealing the immune-modulatory function of the AR pathway. We have described the activation of NK cells by AR inhibitors and the potential synergistic effect with anti-NKG2a combination.1 Strategies to activate NK cells can be limited by the expression of HLA-E, a ligand of the inhibitory checkpoints on NK cells NKG2A. We investigated the mechanisms of AR-dependent modulation of HLA-E on tumor cells and the ADT-enhancing effect on patient-derived NK cells. Methods: PC cell lines were treated with second-generation androgen pathway inhibitors in vitro (enzalutamide 10uM, darolutamide 15 uM), and the expression of HLA-E was evaluated by flow cytometry. To evaluate the modulation of HLA-E by AR, the AR-negative cell lines (PC3 and DU145) were stably transduced with an inducible AR system. The pan HDAC inhibitors vorinostat (0.4 uM) and panobinostat (2.5 uM) were used to evaluate the regulation of HLA-E by epigenetics. We analyzed the activation status of paired peripheral blood NK cells isolated from patients with PCa prior to and post-androgen deprivation therapy (ADT). The patients (n=6) had a median time between collection of 26.3±2.3 days. Results: The AR-responsive LNCaP cell line displayed an increase in surface expression of HLA-E upon treatment with enzalutamide (Enza) or darolutamide (Daro) ([C]: 12.1±1.3%, Enza: 22.3±3.3%, Daro: 19.1±1.1%, p=0.004). AR blockade did not change the surface expression of HLA-E of AR negative PC3 ([C]: 10.5±2.7%, Daro: 13.1±2.3%, p=0.17) or DU145 cell lines ([C]: 3.5±1.7%, Daro: 2.5±0.6%, p=0.12). Transduction of AR in PC3 and DU145 lead to upregulation of HLA-E expression with AR blockade (PC3-AR+ [C]: 13.1±3.7%, Daro: 43.1±5.3%, DU145-AR+ [C]: 4.5±1.4%, Daro: 27.2±3.6%, p=0.001). The upregulation of HLA-E upon AR blockade was suppressed if cell lines were co-treated with vorinostat or panobinostat. Patient-derived peripheral blood NK cells displayed enhanced cytotoxic activity after ADT (expression of Granzyme B pre-ADT: 12.3±2.3%, post-ADT: 36.5±5.7% p=0.0017, Perforin pre-ADT: 3±0.6%, post-ADT: 26.5±3.7% p=0.001), and upregulation of the inhibitory checkpoint NKG2a (pre-ADT: 5.2±1.2%, post-ADT: 12.3±1.4%, p=0.04). Conclusions: Androgen inhibitors upregulate the expression of HLA-E in PCa cell lines by an AR-dependent mechanism regulated by epigenetics. ADT promotes peripheral blood patient-derived NK cell activation and upregulation of inhibitory NKG2A receptor. These findings support further investigative approaches targeting the HLA-E and NKG2A in mCRPC. 1. Schwermann, AACR 2023.

Exhausted signature and regulatory network of NK cells in myasthenia gravis.

NK cells are dysfunctional in myasthenia gravis (MG), but the mechanism is unclear. This study aims to measure associations and underlying mechanisms between the NK cells and the development of MG. Twenty healthy controls (HCs) and 53 MG patients who did not receive glucocorticoids and immunosuppressants were collected. According to the Myasthenia Gravis Foundation of America (MGFA) classification, MG patients were categorized into MGFA I group (n = 18) and MGFA II-IV group (n = 35). Flow cytometry, cell sorting, ELISA, mRNA-sequencing, RT-qPCR, western blot, and cell culture experiments were performed to evaluate the regulatory mechanism of exhausted NK cells. Peripheral NK cells in MGFA II-IV patients exhibit exhausted phenotypes than HCs, marked by the dramatic loss of total NK cells, CD56dimCD16- NK cells, elevated PD1 expression, reduced NKG2D expression, impaired cytotoxic activity (perforin, granzyme B, CD107a) and cytokine secretion (IFN-γ). Plasma IL-6 and IL-21 are elevated in MG patients and mainly derived from the aberrant expansion of monocytes and Tfh cells, respectively. IL-6/IL-21 cooperatively induced NK-cell exhausted signature via upregulating SOCS2 and inhibiting the phosphorylation of STAT5. SOCS2 siRNA and IL-2 supplement attenuated the IL-6/IL-21-mediated alteration of NK-cell phenotypes and function. Inhibition of IL-6/IL-21/SOCS2/STAT5 pathway and recovery of NK-cell ability to inhibit autoimmunity may be a new direction in the treatment of MG.

CD44v6 specific CAR-NK cells for targeted immunotherapy of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major challenge for current therapies. CAR-T cells have shown promising results in blood cancers, however, their effectiveness against solid tumors remains a hurdle. Recently, CD44v6-directed CAR-T cells demonstrated efficacy in controlling tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. Apart from CAR-T cells, CAR-NK cells offer a safe and allogenic alternative to autologous CAR-T cell therapy. In this paper, we investigated the capacity of CAR-NK cells redirected against CD44v6 to execute cytotoxicity against HNSCC. Anti-CD44v6 CAR-NK cells were generated from healthy donor peripheral blood-derived NK cells using gamma retroviral vectors (gRVs). The NK cell transduction was optimized by exploring virus envelope proteins derived from the baboon endogenous virus envelope (BaEV), feline leukemia virus (FeLV, termed RD114-TR) and gibbon ape leukemia virus (GaLV), respectively. BaEV pseudotyped gRVs induced the highest transduction rate compared to RD114-TR and GaLV envelopes as measured by EGFP and surface CAR expression of transduced NK cells. CAR-NK cells showed a two- to threefold increase in killing efficacy against various HNSCC cell lines compared to unmodified, cytokine-expanded primary NK cells. Anti-CD44v6 CAR-NK cells were effective in eliminating tumor cell lines with high and low CD44v6 expression levels. Overall, the improved cytotoxicity of CAR-NK cells holds promise for a therapeutic option for the treatment of HNSCC. However, further preclinical trials are necessary to test in vivo efficacy and safety, as well to optimize the treatment regimen of anti-CD44v6 CAR-NK cells against solid tumors.

Modakafusp Alfa Demonstrates Type I Interferon-Mediated Innate and Adaptive Immune Enhancement in a Phase 1/2 Study in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Modakafusp alfa (moda), a novel immunocytokine, is an innate immunity enhancer comprising two attenuated interferon (IFN) α2b molecules fused to an anti-CD38 IgG4 monoclonal antibody backbone, driving preferential IFNα signalling in CD38-expressing innate and adaptive immune cells as well as myeloma cells. Here, we present pharmacodynamic (PD) data from the first-in-human Phase 1/2 study of moda as a monotherapy in patients with RRMM (iinnovate-1; NCT03215030). A total of 37 patients treated with moda at 1.5 or 3 mg/kg every 4 weeks were included in the analysis. Peripheral blood (PB) and bone marrow (BM) samples were collected during screening or pre-dose on cycle 1 day 1 (C1D1) and at multiple timepoints after treatment. BM and PB samples were evaluated by cytometry time of flight (CyTOF) and bulk RNA sequencing (RNAseq) to assess pharmacodynamic changes in immune cell populations, cell activation, and gene expression. Administration of moda led to enhanced innate immune cell activation and cytotoxic function as demonstrated by increased proportions of CD69+ and granzyme B+ in peripheral NK cells analyzed by CyTOF. This was accompanied by a decrease in the proportion of TIGIT+ NK cells, indicating reduced inhibitory signaling. Furthermore, enhanced proliferation (%Ki67+) of NK cells was observed in both BM and PB. Moda also impacted myeloid cell populations in BM and PB. Dendritic cells (DCs) and monocytes in PB showed increased surface expression of the co-stimulatory molecule CD86, suggesting a potential for enhanced antigen presentation and co-stimulation ability. Increased proliferation (%Ki67) of DCs and monocytes was detected in both PB and BM, accompanied by a decrease in numbers of peripheral DCs which may indicate recruitment or homing to secondary lymphoid organs. Additionally, RNAseq analysis revealed that treatment with moda led to upregulation of CD68 expression in both PB and BM, indicating a proinflammatory M1 macrophage type response. Finally, CyTOF analysis also indicated that moda significantly impacted adaptive immunity. We observed enhanced activation (%CD69, %PD1 and %CD40L) and cytotoxic function (%granzyme B) of peripheral CD8 T cells, and increased CD8 T cell proliferation (%Ki67 CD8) in both PB and BM samples. Enhanced activation of peripheral CD4 T cells (%CD69, %CD40L) was also detected. Consistent with activation of the adaptive immune system, there was a reduction in naïve CD8 T cells accompanied by an increase in CD8 T effector memory cells in PB. Analysis of exhaustion markers (TIGIT, PD-1) in cycle 1 (C1D8, C1D15, and C2D1 predose) showed that moda did not induce CD8 T cell exhaustion in either PB or BM, and there was no change in FoxP3+ Treg cells. Moda-induced innate and adaptive immune cell phenotypic changes were evaluated for correlation with clinical response at timepoints with sufficient number of evaluable samples. While none of the associations were statistically significant after correcting for multiplicity testing (using the false discovery rate method), we observed trends warranting further investigation. There was a greater increase of CD40L+ NK cells and PD1+ CD8 cells in responders vs. non-responders at C1D2. Additionally, at C1D15 responders showed a lower increase in proliferation (%Ki67+) of classical monocytes, NK cells, and myeloid DCs in PBas compared to non-responders. A similar trend was also observed in the BM classical monocytes. Even though the peak of Ki67 expression was observed at C1D8, diminished increases of proliferating cells at C1D15 in responders was a surprising observation that needs further consideration. The PD biomarker data from this first-in-human clinical trial demonstrated that treatment with moda enhances both innate and adaptive immune cell activation in PB and BM. Importantly, moda-mediated immune activation did not result in T cell exhaustion during cycle 1, as evaluated in both PB and BM. We have observed interesting trends of immune phenotypic changes that may correlate with response. Follow-up analyses of the correlation of biomarkers with clinical response are in progress and will be presented. Further clinical trials are underway (iinnovate-2, NCT05556616; iinnovate-3, NCT05590377) to evaluate moda's novel immune activating mechanism in combination with standard of care anti-myeloma therapies.

Dynamic immune cell profiling identified natural killer cell shift as the key event in early allograft dysfunction after liver transplantation.

Early allograft dysfunction (EAD) is a life-threatening and fast-developing complication after liver transplantation. The underlying mechanism needs to be better understood, and there has yet to be an efficient therapeutic target. This study retrospectively reviewed 109 patients undergoing liver transplantation, with dynamic profiling of CD3/4/8/16/19/45/56 on the peripheral immune cells (before transplant and 2-4 days after). Altogether, 35 out of the 109 patients developed EAD after liver transplantation. We observed a significant decrease in the natural killer cell proportion (NK cell shift, p = 0.008). The NK cell shift was linearly correlated with cold ischemic time (p = 0.016) and was potentially related to the recipients' outcomes. In mouse models, ischemic/reperfusion (I/R) treatments induced the recruitment of NK cells from peripheral blood into liver tissues. NK cell depletion blocked a series of immune cascades (including CD8+ CD127+ T cells) and inhibited hepatocyte injury effectively in I/R and liver transplantation models. We further found that I/R treatment increased hepatic expression of the ligands for natural killer group 2 member D (NKG2D), a primary activating cell surface receptor in NK cells. Blockade of NKG2D showed a similar protective effect against I/R injury, indicating its role in NK cell activation and the subsequent immunological injury. Our findings built a bridge for the translation from innate immune response to EAD at the bedside. Peripheral NK cell shift is associated with the incidence of EAD after liver transplantation. NKG2D-mediated NK cell activation is a potential therapeutic target.

Features of NK cell phenotype virus genotype-driven chronic viral hepatitis C

Elimination of the hepatitis C virus (HCV) due to direct antiviral drug (DAD) action affects alteration in virus phenotype and, accordingly, NK cell functional activity. However, the published data are very contradictory. The aim of the study was to investigae alterations in NK cell subset phenotype after DAD treatment of HCV genotype-dependent chronic viral hepatitis C (CVHC) patients. Materials and methods. 111 CVHC patients and 21 healthy volunteers were examined. The diagnosis was established on epidemiological, clinical and laboratory data. All 111 subjects with CVHC received direct antiviral drugs Sofosbuvir and Velpatasvir for 12 weeks. The study of the NK cell phenotypes wwas analyzed by multicolor flow cytometry. Results. A decreased count of cytokine-producing along with increased frequency of cytotoxic NK cells were found in CVHC patients blood samples with various HCV genotypes prior to DAD treatment. The imbalance of cytotoxic cells with a high level of functional activity was also found in CVHC patients regardless of HCV genotype. The patients with HCV genotypes 1 and 3 showed significantly increased level of immunoregulatory NK cells. In addition, increased count of glycohydrolase (CD38) and ecto-5'-nucleotidase (CD73)-expressing NK cells were found in patients with HCV genotypes 1 and 3. Hence, such alterations in NK cell phenotype in CVHC patients were presented as sustained high viral load which peaking at carriers of HCV genotype 1 that was minimal in patients with HCV genotype 2. The most prominent change in NK cells after DAD treatment was found in CVHC patients with HCV genotype 2 (normalization of CD8-expressing NK cell subset composition and count). Only patients with HCV genotype 2 after treatment had increased frequencies of peripheral blood double-negative CD38–CD73– NK cells. Patients with HCV genotypes 1 and 3 also showed minimally improved in NK cell subset composition after DAD treatment. Conclusion. Evaluation of specific changes in NK cell phenotype during DAD treatment of CVHC patients driven by HCV genotype undoubtedly is of importance and high relevance. The results obtained are novel and complement the insights into CVHC immunopathogenesis. Analysis of NK cell phenotypes and functional activity in patients with CVHC may promote development of new methods for treating HCV infection.

Pediatric IBD patients show medication and disease activity dependent changes in NK cell and CD4 memory T cell populations.

CD4+ memory T cells facilitate long-termed adaptive immune responses while NK cells are predominately rapid effector cells with significant functions for both intestinal homeostasis and inflammation. We wanted to study both populations in health and pediatric inflammatory bowel disease (IBD) and correlate them with disease activity and medication. We performed flow cytometric analyses of peripheral blood CD4 + CD45RO+ memory T cells and CD3-CD16 + CD56+ NK cells in 30 patients with IBD and 31 age-matched controls and correlated percentages of subsets with disease activity (PUCAI/PCDAI) and medication. We found a significant reduction of peripheral NK cells in overall IBD patients with both clinical remission and disease activity, which was even more pronounced in patients treated with azathioprine. Otherwise, circulating CD4+ memory T cell populations were significantly enhanced in active IBD compared to controls. Enhancement of memory T cells was particularly found in new onset disease and correlated with disease activity scores. Our single center cohort confirms previous results showing enhanced memory T cell populations in pediatric IBD patients, which correlate with disease activity scores. CD4+ memory T cells are a relevant pathogenic leukocyte population for disease development and perpetuation in IBD. In addition, we found a decrease of NK cells in IBD patients, which was pronounced by use of azathioprine. Surveillance of both cellular populations could possibly serve as biomarker for therapy control in pediatric IBD.

P-339 Activities and changes in the number of natural killer cells in endometriosis: systematic review and meta-analysis

Abstract Study question What are the differences in peripheral, uterine, and peritoneal NK (uNK, pNK, pfNK) cell counts/percentages, and activities in women with endometriosis compared controls Summary answer The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference in women with endometriosis compared controls What is known already NK cells play an important role in the pathogenesis of endometriosis Study design, size, duration This systematic review and meta-analysis included 36 experimental studies (case control and cross sectional). Narrative review is also conducted on the NK cell activity, cytokine expression, regulations and receptors. Participants/materials, setting, methods Women suffering with endometriosis confirmed with laparoscopy and/or pathology was the cases whereas, women having different pelvic pathology such as myoma, ovarian cyst, healthy woman and verified not having endometriosis with laparoscopy and/or pathology was considered as controls for this study. Databases (PubMed, Web of Science, Scopus, Google scholar, and EMBASE through OVID) were used to search for the available studies. RevMan 5.4 and STATA 14 were used to analyze the data. Main results and the role of chance The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference (standard mean difference SMD 0.13, 95%CI -0.35, 0.62; P = 0.59, I2 74%; total 393 women, 6 studies), (SMD 0.27, 95%CI -0.07, 0.61; P = 0.12, I2 80%; total 825 women, 11 studies), and (SMD 0.31, 95%CI -0.68, 01.29; P = 0.59, I2 94%; total 404 women,7 studies) in endometriosis patients compared with controls respectively. The pooled NK (uNK, pNK, pfNK) cell cytotoxicity/activity level is significantly higher in controls compared with women with endometriosis (MD 5.43, 95%CI 2.29, 8.57; P &amp;lt; 0.007, I2 50%; total 323 women, 7 studies). The NK cytotoxicity/activity level in early stages of endometriosis has no significant difference compared with advanced stages of endometriosis (MD 2.29, 95%CI –1.98-6.57; P = 0.29, I2 87%; total 263 women, 7 studies). There are variations in studies conducted in NK cell activities in endometriosis: broadly categorized as NK cell cytotoxicity, cytokine expression and NK cell regulation and receptors (Inhibition and activation). The cytotoxic activity of NK cells decreased in women with endometriosis and correlated with the severity of the disease, however the cytokine expression and inhibitor receptors are equivocal across studies. Limitations, reasons for caution We unable to conduct sub-group analysis to investigate the cytotoxic activity of NK cell in women with endometriosis and its stages in different samples such as pNK, uNK, and pfNK due to the lack of studies for the analysis. Wider implications of the findings This study revealed that the activity of NK cells in women with endometriosis is significantly lower than controls.Though the mean difference of NK cell count and percentage has no significant difference, the cytotoxicity activity of NK cell is higher in controls compared with women with endometriosis Trial registration number CRD42022384060

Clinical safety and efficacy observation of ex vivo-expanded peripheral blood-derived allogeneic NK cells in the treatment of refractory or recurrent ovarian cancer.

e17553 Background: Ovarian cancer is the most lethal gynecologic malignancy. Nearly 75% of patients were diagnosed at advanced stages. Despite 60%-80% of patients achieve complete remission after treatment with surgery combined with chemotherapy, about 60% of patients relapse within two years, more than 50% of patients develop chemotherapy tolerance with only 30% five-year survival rate. Platinum resistance-induced recurrence and platinum intolerance result in poor prognosis. Thus, there is an unmet need for innovative therapy of advanced ovarian cancer. We have developed a novel non-genetically modified ex vivo-expanded allogeneic NK cells from peripheral blood (PB). The expanded PB-NK cells have highly cytolytic activity and have the capacity to preferentially reside in ovary. We conducted a phase I clinical trial for evaluation of safety and efficacy of allogeneic NK cells in treatment of patients with refractory or recurrent ovarian cancers. Methods: Patients with advanced ovarian cancer who failed after multiline treatment were enrolled and received the treatment with allogeneic NK cells. The treatment was divided into low, medium and high dose groups with a 28-day cycle. safety and efficacy were evaluated after receiving two cycles of allogeneic NK cell infusion. Results: A total of 4 patients with advanced ovarian cancer who failed multiline therapy completed two courses of cell infusion therapy and evaluation. The median age of patients was 58 years (range 50 to 71 years). The median number of lines of treatment was 6 (range 3-7). One recurrent patient with platinum intolerance received low dose of NK cells and three relapse or refractory patients with platinum resistance received medium dose of allogeneic NK cell therapy. The patient in low-dose group and one patient in medium-dose group were evaluated for stable disease (SD), the other two patients in medium-dose group were disease progression after two courses of treatment. Notable, the patient in low-dose group continued to receive treatment with the same dose and achieved partial response (PR) after three courses of treatment. Upon receiving continuous NK cell therapy, the tumor lesions continued to shrink, with the best result was 60% reduction. It was re-evaluated as progressive disease (PD) 6 months after treatment was discontinued. Adverse events during the follow-up period included grade I fever in 4 (100%), grade I drowsiness in 1 (25%), grade I chills in 1 (25%), and grade I malaise in 1 (25%). No CRS or GVHD occurred. Conclusions: We report the safety and efficacy of allogeneic PB-NK cells in treatment of relapse or refractory ovarian cancer. The results showed that the allogeneic PB-NK cells are safe and feasible for the treatment of ovarian cancer, indicating a promising curative effect. There is no host versus graft reaction upon receiving continuous allogeneic NK cell therapy. Clinical trial information: NCT03619954 .

FRI-366 - Cytotoxic activity of peripheral NK cells is decreased in primary sclerosing cholangitis

FRI-366 - Cytotoxic activity of peripheral NK cells is decreased in primary sclerosing cholangitis

Abstract CT021: Assessing clinical and pharmacodynamic (PD) profiles of patients (pts) with chronic lymphocytic leukemia (CLL) on ianalumab (VAY736) + ibrutinib

Abstract Introduction VAY736 is an afucosylated, human monoclonal antibody engineered to enhance antibody-dependent cellular cytotoxicity that targets BAFF-R+ B cells for elimination. In preclinical CLL models, VAY736 showed antileukemic activity and, when combined with ibrutinib, significantly reduced disease burden, which may allow some pts to discontinue ibrutinib. Methods This Phase Ib dose-escalation (ESC)/-expansion (EXP) trial (NCT03400176) enrolled pts with CLL who did not achieve a complete response (CR) after &amp;gt;1 year of ibrutinib or had developed a resistance mutation to ibrutinib. Pts received IV VAY736 (ESC: 0.3-9 mg/kg; EXP: 3 mg/kg) once every 2 weeks and oral ibrutinib (420 mg) once daily for up to 8 28-day cycles. Pts achieving undetectable MRD (uMRD) at C9D1 could discontinue ibrutinib at investigator discretion. The study aimed to characterize the safety and tolerability of VAY736 + ibrutinib, assess antitumor activity, PK, and characterize PD profiles. Results By Jul 29, 2022, 39 pts were enrolled (ESC: n=15; EXP: n=24). Table 1 shows pt characteristics, safety, and efficacy data. The overall response at C9D1 for 37 evaluable pts was 40.5% CR + CRi and 16.2% PR (1L: 63.6% CR + CRi and 18.2% PR). At C9D1, 17 pts (45.9%) had uMRD in blood or bone marrow (BM). In the 2-year follow-up period, 16 pts discontinued ibrutinib and were off therapy for 4.9-19.8 months. Frequency of peripheral NKp46+ NK cells increased at least 50% after VAY736 in over 50% of pts. Preliminary coverage-based limiting-cell experiment analysis of RNAseq (CLEAR) data from 10 pts supports peripheral NK cell activation with VAY736. Conclusions VAY736 + ibrutinib appears highly active and has an acceptable safety profile. Multiple pts attained uMRD in blood or BM. Biomarker data suggest NK cell activation with VAY736. More pts will be included in the RNAseq analysis at presentation. Future development of VAY736 for CLL is strongly indicated based on these promising data. Table 1. Patient characteristics, safety, and efficacy results. All patients (N=39) Patient demographics and prior treatment Median age, years (range) 65.0 (39-82) ECOG performance status, n (%) 0 36 (92.3) 1 3 (7.7) No prior regimens excluding ibrutinib, n (%) 12 (30.8) Median number of prior regimens, n (range) 1.0 (0.0-14.0) Median duration of ibrutinib, years (range) 2.95 (0.2-8.3) Patient baseline characteristics Dohner risk by FISH,a n (%) 17p deletion 6 (15.4) 11q deletion 9 (23.1) Trisomy 12 3 (7.7) 13q deletion 10 (25.6) IGHV mutant status, n (%) Non-mutant 32 (82.1) Complex karyotype, n (%) Yes 20 (51.3) Safety Dose-limiting toxicities, n (%) 0 Patients with at least one AE, any grade, n (%) 38 (97.4) Patients with at least one Grade ≥3 AE, n (%) 13 (33.3) Most common (occurring in ≥2 patients) Grade ≥3 AEs, n (%) Neutrophil count decreased 5 (12.8) Lymphocyte count decreased 2 (5.1) Hypophosphatemia 2 (5.1) Lipase increased 2 (5.1) Efficacy 1Lb n=11 R/R n=26 Evaluable patients N=37 Overall response at C9D1 or before discontinuation,c n (%) Complete response 6 (54.5) 8 (30.8) 14 (37.8) Complete response with incomplete marrow recovery 1 (9.1) 0 1 (2.7) Partial response 2 (18.2) 4 (15.4) 6 (16.2) Stable disease 2 (18.2) 8 (30.8) 10 (27.0) Progressive disease 0 5 (19.2) 5 (13.5) uMRD response at C9D1 or before discontinuation,c n (%) Bone marrow uMRD 6 (54.5) 6 (23.1) 12 (32.4) Blood uMRD 7 (63.6) 10 (38.5) 17 (45.9) Blood or bone marrow uMRD 7 (63.6) 10 (38.5) 17 (45.9) Patients elected to discontinue ibrutinib after achieving CR or uMRD, n (%) 16 (43.2) aThe categories were: patients with a 17p deletion; patients with an 11q deletion without a 17p deletion; patients with trisomy 12 without a 17p deletion or an 11q deletion; and patients with a 13q deletion without a 17p deletion, trisomy 12, or an 11q deletion; bPatients with no prior therapies excluding ibrutinib; cFor evaluable patients (N=37). 1L, first line; AE, adverse event; CR, complete response; C, cycle; D, day; ECOG, Eastern Cooperative Oncology Group; FISH, fluorescence in situ hybridization; IGHV, immunoglobulin heavy chain variable region; R/R, relapsed/refractory; uMRD, undetectable minimal residual disease. Citation Format: Kerry Anne Rogers, Pearlly Yan, Ian W. Flinn, Deborah M. Stephens, Thomas J. Kipps, Sarah M. Larson, Laura Martz, Xi Chen, Huabao Wang, Ethan Hopping, Ralf Bundschuh, Alexandra Acosta, Daniela Baldoni, Anwesha Chaudhury, Jeanne Whalen, Nadia B. Hassounah, Nina Orwitz, Janghee Woo, John C. Byrd. Assessing clinical and pharmacodynamic (PD) profiles of patients (pts) with chronic lymphocytic leukemia (CLL) on ianalumab (VAY736) + ibrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT021.