Peripheral Blood Natural Killer Cells Research Articles

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Natural Killer Different Subpopulations of Peripheral Blood During Pregnancy

The functions of peripheral blood NK cells change significantly during pregnancy, which is mainly due to the inhibition of their cytotoxicity. The functional activity of NK cells is directly related to their metabolic status, but these changes in physiological pregnancy have not been studied. The aim of this work is to study the expression of Glut-1, CD94 and CD107a molecules characterizing metabolic and cytotoxic activity, as well as the mitochondrial mass of different subpopulations of peripheral blood NK cells in the I and III trimesters of physiological pregnancy. The object of the study was the peripheral blood of healthy women in the I and III trimesters of physiological pregnancy. The control group consisted of healthy non-pregnant women in the follicular phase of the menstrual cycle. The expression of Glut-1, CD94, CD107a molecules and the mitochondrial mass were analyzed by flow cytometry on regulatory (CD16–CD56bright), cytotoxic (CD16+CD56dim), minor cytotoxic (CD16hiCD56–) NK cells. It was found that in non-pregnant women, minor cytotoxic CD16hiCD56–NK have the highest expression of Glut-1, CD107a and the lowest expression of CD94 compared to other NK cell subpopulations. On regulatory CD16–CD 56bright and cytotoxic CD16+CD56dimNK, the expression of these molecules is comparable to each other. The mitochondrial mass is similar in all studied subpopulations. In the first trimester, the expression of Glut-1 increases on regulatory CD16–CD56brightNK, the mitochondrial mass and the expression of CD94, CD107a in all NK cells do not differ from non-pregnant ones. In the third trimester, the mitochondrial mass increases in cytotoxic CD16+CD56dimNK cells, but CD94 expression decreases compared to non-pregnant ones, and the expression CD94 in regulatory CD16–CD56brightNK increases compared to the first trimester. CD107a expression in minor cytotoxic CD16hiCD56–NK decreases, but in other subpopulations does not change compared to non-pregnant. The expression of Glut-1 does not change in all subpopulations. Thus, different subpopulations of peripheral blood NK cells are heterogeneous in the expression of Glut-1, CD107a, CD94. The expression of these molecules during physiological pregnancy varies by trimester. The obtained results are important for understanding the mechanisms of NK cell function regulations during pregnancy.

An altered natural killer cell immunophenotype characterizes clinically severe pediatric RSV infection.

Respiratory syncytial virus (RSV) infects nearly all children by 2 years of age and is a leading cause of pediatric hospitalizations. A subset of children with RSV infection (RSV+ children) develop respiratory failure requiring intensive care, but immune mechanisms distinguishing severe pediatric RSV infection are not fully elucidated. Natural killer (NK) cells are key innate immune effectors of viral host defense. In this study of 47 critically ill RSV+ children, we coupled NK cell immunophenotype and cytotoxic function with clinical parameters to identify an NK cell immune signature of severe pediatric RSV disease. Airway NK cells were increased in intubated RSV+ children with severe hypoxemia and prolonged duration of mechanical ventilation and were correlated with clinical severity scores. Peripheral blood NK cells were decreased in RSV+ patients and had altered activating receptor expression, with increased expression of CD69 and decreased expression of NKG2D. Ex vivo, circulating NK cells from RSV+ patients exhibited functional impairment characterized by decreased cytotoxicity as well as aberrant immune synapse assembly and lytic granule trafficking. NK cell frequency and phenotype correlated with clinical measures that defined disease severity. These findings implicate a role for NK cells in mediating RSV immunopathology and suggest that an altered NK cell immunophenotype is associated with severe RSV disease in young children.

Tumor Resection in Hepatic Carcinomas Restores Circulating T Regulatory Cells.

Background/Objectives: Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) represent major primary liver cancers, affecting one of the most vital organs in the human body. T regulatory (Treg) cells play an important role in liver cancers through the immunosuppression of antitumor immune responses. The current study focuses on the characterization of circulating natural killer (NK) cells and T cell subsets, including Treg cells, in CCA and HCC patients, before and after surgical tumor resection, in order to understand the effect of tumor resection on the homeostasis of peripheral blood NK cells and T cells. Methods: Whole blood assays were performed to monitor immune alterations and the functional competence of circulating lymphocytes in a group of ten healthy individuals, eight CCA patients, and twenty HCC patients, before and one month after the surgical procedure, using flow cytometry, cell sorting, and qRT-PCR. Results: Before tumor resection, both HCC and CCA patients display increased percentages of CD8+ Treg cells and decreased frequencies of circulating CD4+ Treg cells. Notwithstanding, no functional impairment was detected on circulating CD4+ Treg cells, neither in CCA nor in HCC patients. Interestingly, the frequency of peripheral CD4+ Treg cells increased from 0.55% ± 0.49 and 0.71% ± 0.54 (in CCA and HCC, respectively) at T0 to 0.99% ± 0.91 and 1.17% ± 0.33 (in CCA and HCC, respectively) at T1, following tumor resection. Conclusions: Our results suggest mechanisms of immune modulation induced by tumor resection.

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.

Chronic lymphoproliferative disorder of natural killer cells-related neurolymphomatosis with severe autonomic dysfunction: a case report

BackgroundChronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a rare disease characterized by a persistent increase in NK cells in peripheral blood and is generally asymptomatic. If present, symptoms may include fatigue, B symptoms (fever, night sweats, and unintentional weight loss), autoimmune-associated diseases, splenomegaly, and infection due to neutropenia. Peripheral neuropathy, however, is uncommon with an incidence of 3%. Neurolymphomatosis is a neurological manifestation of non-Hodgkin lymphoma and leukemia in which neurotropic neoplastic cells infiltrate the nerves. Moreover, neurolymphomatosis caused by CLPD-NK is extremely rare, with even fewer cases of autonomic dysfunction. We report a case of neurolymphomatosis associated with CLPD-NK and developed autonomic dysfunction, including orthostatic hypotension and gastrointestinal symptoms.Case presentationThe patient was a 61-year-old male who was referred to our hospital for leukocytosis. He was diagnosed with CLPD-NK; however, was untreated since he had no hepatosplenomegaly, and other systemic symptoms. He later developed numbness in his lower extremities. Cerebral spinal fluid examination revealed a markedly elevated protein level of 140 mg/dL, and contrast-enhanced magnetic resonance imaging showed bilateral L4 and 5 nerve roots with enlargement and contrast effect. An immune-mediated polyradiculoneuropathy was suspected, and he was treated with intravenous methylprednisolone and immunoglobulin followed by oral prednisolone and cyclosporine. Although his symptoms were relieved by the immunotherapy, significant autonomic dysfunction, including intractable diarrhea, decreased sweating, and orthostatic hypotension, appeared. Additionally, tests for onconeuronal antibodies, ganglionic nicotinic acetylcholine receptor (gAChR) antibody, NF155, CNTN1, Caspr1 antibody, and anti-ganglioside antibodies were all negative. A sural nerve biopsy revealed lymphocytic infiltration, and immunohistochemical staining of lymphocytes confirmed the infiltration of NK and T cells. Therefore, a diagnosis of neurolymphomatosis caused by CLPD-NK was made, and chemotherapy led to partial symptom improvement.ConclusionsWe experienced a case of pathologically diagnosed neurolymphomatosis with autonomic dysfunction associated with CLPD-NK. In cases of subacute to chronic autonomic dysfunction, paraneoplastic neuropathy, amyloidosis, and autoimmune autonomic ganglionopathy are considered; however neurolymphomatosis caused by CLPD-NK, an important cause of autonomic dysfunction, is not. In difficult to make diagnosis, aggressive nerve biopsy is required.

HBcrAg values may predict virological and immunological responses to pegIFN-α in NUC-suppressed HBeAg-negative chronic hepatitis B

ObjectiveSelected populations of patients with chronic hepatitis B (CHB) may benefit from a combined use of pegylated interferon-alpha (pegIFN-α) and nucleos(t)ides (NUCs). The aim of our study was to assess...

Proliferation capability of natural killer cells upon cytokines stimulation correlated negatively with serum lactate dehydrogenase level in coronary artery disease patients.

Natural killer (NK) cells are proposed to participate in coronary artery disease (CAD) development. However, little is known about how CAD patients' NK cells respond to different stimulatory factors in terms of proliferation capability. Twenty-nine CAD patients' peripheral blood NK cells were isolated and individually treated with IL-2, IL-12, IL-15, IL-18, IL-21, cortisone acetate, hydrocortisone, or ascorbic acid for 36 hours, followed by cell cycle analysis using flow cytometry. The ratio of S and G2/M phase cell number to total cell number was defined as a proliferation index (PrI) and used for proliferative capability indication. The results showed that these eight factors resulted in different life cycle changes in the 29 NK cell samples. Remarkably, 28 out of 29 NK cell samples showed an obvious increase in PrI upon ascorbic acid treatment. The serum lactate dehydrogenase (LDH) level of the 29 CAD patients was measured. The results showed a negative correlation between serum LDH level and the CAD patients' NK cell PrI upon stimulation of interleukins, but not the non-interleukin stimulators. Consistently, a retrospective analysis of 46 CAD patients and 32 healthy donors showed that the circulating NK cell number negatively correlated with the serum LDH level in CAD patients. Unexpectedly, addition of LDH to NK cells significantly enhanced the production of IFN-γ, IL-10 and TNF-α, suggesting a strong regulatory role on NK cell's function. Ascorbic acid could promote the proliferation of the CAD patients' NK cells; LDH serum level may function as an indicator for NK cell proliferation capability and an immune-regulatory factor.

Potential role of B- and NK-cells in the pathogenesis of pediatric aplastic anemia through deep phenotyping.

Pediatric patients with unexplained bone marrow failure (BMF) are often categorized as aplastic anemia (AA). Based on the accepted hypothesis of an auto-immune mechanism underlying AA, immune suppressive therapy (IST) might be effective. However, due to the lack of diagnostic tools to identify immune AA and prognostic markers to predict IST response together with the unequaled curative potential of hematopoietic stem cell transplantation (HSCT), most pediatric severe AA patients are momentarily treated by HSCT if available. Although several studies indicate oligoclonal T-cells with cytotoxic activities towards the hematopoietic stem cells, increasing evidence points towards defective inhibitory mechanisms failing to inhibit auto-reactive T-cells. We aimed to investigate the role of NK- and B-cells in seven pediatric AA patients through a comprehensive analysis of paired bone marrow and peripheral blood samples with spectral flow cytometry in comparison to healthy age-matched bone marrow donors. We observed a reduced absolute number of NK-cells in peripheral blood of AA patients with a skewed distribution towards CD56bright NK-cells in a subgroup of patients. The enriched CD56bright NK-cells had a lower expression of CD45RA and TIGIT and a higher expression of CD16, compared to healthy donors. Functional analysis revealed no differences in degranulation. However, IFN-γ production and perforin expression of NK-cells were reduced in the CD56bright-enriched patient group. The diminished NK-cell function in this subgroup might underly the auto-immunity. Importantly, NK-function of AA patients with reduced CD56bright NK-cells was comparable to healthy donors. Also, B-cell counts were lower in AA patients. Subset analysis revealed a trend towards reduction of transitional B-cells in both absolute and relative numbers compared to healthy controls. As these cells were previously hypothesized as regulatory cells in AA, decreased numbers might be involved in defective inhibition of auto-reactive T-cells. Interestingly, even in patients with normal distribution of precursor B-cells, the transitional compartment was reduced, indicating partial differentiation failure from immature to transitional B-cells or a selective loss. Our findings provide a base for future studies to unravel the role of transitional B-cells and CD56bright NK-cells in larger cohorts of pediatric AA patients as diagnostic markers for immune AA and targets for therapeutic interventions.

Recombinant Human Interleukin-2 Corrects NK Cell Phenotype and Functional Activity in Patients with Post-COVID Syndrome

Post-COVID syndrome develops in 10–20% of people who have recovered from COVID-19 and it is characterized by impaired function of the nervous, cardiovascular, and immune systems. Previously, it was found that patients who recovered from infection with the SARS-CoV-2 virus had a decrease in the number and functional activity of NK cells. The aim of the study was to assess the effectiveness of recombinant human IL-2 (rhIL-2) administered to correct NK cell phenotype and functional activity in patients with post-COVID syndrome. Patients were examined after 3 months for acute COVID-19 of varying severity. The phenotype of the peripheral blood NK cells was studied by flow cytometry. It was found that disturbances in the cell subset composition in patients with post-COVID syndrome were characterized by low levels of mature (p = 0.001) and cytotoxic NK cells (p = 0.013), with increased release of immature NK cells (p = 0.023). Functional deficiency of NK cells in post-COVID syndrome was characterized by lowered cytotoxic activity due to the decreased count of CD57+ (p = 0.001) and CD8+ (p 0.001) NK cells. In the treatment of patients with post-COVID syndrome with recombinant IL-2, peripheral blood NK cell count and functional potential were restored. In general, the effectiveness of using rhIL-2 in treatment of post-COVID syndrome has been proven in patients with low levels of NK cells.

The impact of CLDN18.2 expression on effector cells mediating antibody-dependent cellular cytotoxicity in gastric cancer

Activating antibody-dependent cellular cytotoxicity (ADCC) by targeting claudin-18 isoform 2 (CLDN18.2) using zolbetuximab, a monoclonal antibody against CLDN18.2, has been considered a promising novel therapeutic strategy for gastric cancer (GC). However, the impact of CLDN18.2 expression on natural killer (NK) cells and monocytes/macrophages—crucial effector cells of ADCC—in GC has not been fully investigated. In the present study, we assessed the impact of CLDN18.2 expression on clinical outcomes, molecular features, and the frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, in GC by analyzing our own GC cohorts. The expression of CLDN18.2 did not significantly impact clinical outcomes of GC patients, while it was significantly and positively associated with Epstein–Barr virus (EBV) status and PD-L1 expression. The frequencies of tumor-infiltrating NK cells and macrophages, as well as peripheral blood NK cells and monocytes, were comparable between CLDN18.2-positive and CLDN18.2-negative GCs. Importantly, both CLDN18.2 expression and the number of tumor-infiltrating NK cells were significantly higher in EBV-associated GC compared to other molecular subtypes. Our findings support the effectiveness of zolbetuximab in CLDN18.2-positive GC, and offer a novel insight into the treatment of this cancer type, highlighting its potential effectiveness for CLDN18.2-positive/EBV-associated GC.

More balance toward activating receptors and cytotoxic activity of NK cells ex vivo differentiated from human umbilical cord blood-derived CD34+ stem cells in comparison with peripheral blood NK cells

Adoptive immunotherapies that use functional NK cells depend on the availability of sufficient numbers of these cells. We expanded umbilical cord blood (UCB)-CD34+ HSCs for 2 weeks and then differentiated them into NK cells and compared their function to peripheral blood (PB) NK cells. We assessed NKG2D, NKG2A, NKp30, NKp44, NKp46, and the expression of CD107a, CD57, CD69, FasL, PD-1, and IFN-γ level in two groups after co-culture with K562 cell line. We found that UCB-CD34+-derived NK cells express significantly more NKG2D, NKp44, and NKp46 receptors than PB NK cells. PB NK cells expressed significantly higher NKG2A and CD57 than UCB-CD34+-derived NK cells. In addition, UCB-CD34+-derived NK cells significantly expressed CD107a more than PB NK cells. Based on our findings, UCB-CD34+ cells can be a potentially advantageous source with strong cytotoxic function to produce allogeneic NK cells for adoptive cancer immunotherapy.

Scalable process development of NK and CAR-NK expansion in a closed bioreactor.

Production of large amounts of functional NK and CAR-NK cells represents one of the bottlenecks for NK-based immunotherapy. In this study, we developed a large-scale, reliable, and practicable NK and CAR-NK production using G-Rex 100M bioreactors, which depend on a gas-permeable membrane technology. This system holds large volumes of medium with enhanced oxygen delivery, creating conditions conducive to large-scale PBNK and CAR-NK expansions for cancer therapy. Both peripheral blood NK cells (PBNKs) and CAR-NKs expanded in these bioreactors retained similar immunophenotypes and exhibited comparable cytotoxicity towards hepatocellular carcinoma (HCC) cells akin to that of NK and CAR-NK cells expanded in G-Rex 6 well bioreactors. Importantly, cryopreservation minimally affected the cytotoxicity of NK cells expanded using the G-Rex 100M bioreactors, establishing a robust platform for scaled-up NK and CAR-NK cell production. This method is promising for the development of "off-the-shelf" NK cells, supporting the future clinical implementation of NK cell immunotherapy.

Study on the Standardized Preparation of NK Cells and the Mechanism of Anti-aging of Rhesus Monkey PBMC

Background: Natural killer (NK) cells are an important part of the immune system and play a central role in cell-mediated immune responses. Recent studies have shown that NK cells promote organ rejuvenation, thus achieving anti-aging effects from the inside to out. Methods: We amplified and cultured human peripheral blood NK cells and the positive rate reached 43.91%. After purification, the positive rate reached 92.65%. The cultured NK showed a good killing effect on tumor cells and the NK cells reached the standard preparation scale. The NK cells cultured by us were co-cultured with the PBMC of aging macaques and the quantitative PCR and WB results showed that the expression of aging gene and aging protein of the PBMC of aging macaques decreased significantly after co-culture, with statistical significance (P less than 0.01). Result: These results indicate that NK cells prepared with standard preparation have anti-aging effect, which will have extensive significance in preclinical research.

A novel NKp80-based strategy for universal identification of normal, reactive and tumor/clonal natural killer-cells in blood.

Natural killer (NK) cells are traditionally identified by flow cytometry using a combination of markers (CD16/CD56/CD3), because a specific NK-cell marker is still missing. Here we investigated the utility of CD314, CD335 and NKp80, compared to CD16/CD56/CD3, for more robust identification of NK-cells in human blood, for diagnostic purposes. A total of 156 peripheral blood (PB) samples collected from healthy donors (HD) and patients with diseases frequently associated with loss/downregulation of classical NK-cell markers were immunophenotyped following EuroFlow protocols, aimed at comparing the staining profile of total blood NK-cells for CD314, CD335 and NKp80, and the performance of distinct marker combinations for their accurate identification. NKp80 showed a superior performance (vs. CD314 and CD335) for the identification of NK-cells in HD blood. Besides, NKp80 improved the conventional CD16/CD56/CD3-based strategy to identify PB NK-cells in HD and reactive processes, particularly when combined with CD16 for further accurate NK-cell-subsetting. Although NKp80+CD16 improved the identification of clonal/tumor NK-cells, particularly among CD56- cases (53%), aberrant downregulation of NKp80 was observed in 25% of patients, in whom CD56 was useful as a complementary NK-cell marker. As NKp80 is also expressed on T-cells, we noted increased numbers of NKp80+ cytotoxic T-cells at the more advanced maturation stages, mostly in adults. Here we propose a new robust approach for the identification of PB NK-cells, based on the combination of NKp80 plus CD16. However, in chronic lymphoproliferative disorders of NK-cells, addition of CD56 is recommended to identify clonal NK-cells, due to their frequent aberrant NKp80- phenotype.

Decreased Peripheral Blood Natural Killer Cell Count in Untreated Juvenile Dermatomyositis Is Associated with Muscle Weakness.

Juvenile Dermatomyositis (JDM) is the most common inflammatory myopathy in pediatrics. This study evaluates the role of Natural Killer (NK) cells in Juvenile Dermatomyositis (JDM) pathophysiology. The study included 133 untreated JDM children with an NK cell count evaluation before treatment. NK cell subsets (CD56low/dim vs. CD 56bright) were examined in 9 untreated children. CD56 and perforin were evaluated in situ in six untreated JDM and three orthopedic, pediatric controls. 56% of treatment-naive JDM had reduced circulating NK cell counts, designated "low NK cell". This low NK group had more active muscle disease compared to the normal NK cell group. The percentage of circulating CD56low/dim NK cells was significantly lower in the NK low group than in controls (0.55% vs. 4.6% p < 0.001). Examination of the untreated JDM diagnostic muscle biopsy documented an increased infiltration of CD56 and perforin-positive cells (p = 0.023, p = 0.038, respectively). Treatment-naive JDM with reduced circulating NK cell counts exhibited more muscle weakness and higher levels of serum muscle enzymes. Muscle biopsies from treatment-naive JDM displayed increased NK cell infiltration, with increased CD56 and perforin-positive cells.

Effect of preoperative natural killer cell on postoperative pulmonary complications in patients of lung cancer − A single-center retrospective cohort study

BackgroundThe effect of preoperative natural killer (NK) cell abnormalities on postoperative pulmonary complications (PPCs) after thoracoscopic radical resection of lung cancer is still unclear. The main purpose of this study was to investigate the relationship between the preoperative NK cell ratio and PPCs. MethodsThe patients who underwent thoracoscopic radical resection for lung cancer were divided into a normal group and an abnormal group according to whether the proportion of preoperative NK cells was within the reference range. The main outcome was the incidence of PPCs during postoperative hospitalization. The demographic and perioperative data were collected. Propensity score matching was used to exclude systematic bias. Univariate logistic regression was used to test the relationship between the preoperative NK cell ratio and the incidence of PPCs. The restrictive cubic spline curve was used to analyze the dose–effect relationship between the preoperative NK cell ratio and the incidence of PPCs. ResultsA total of 4161 patients were included. After establishing a matching cohort, 910 patients were included in the statistical analysis. The incidence of PPCs in the abnormal group was greater than that in the normal group (55.2% vs. 31.6%). The incidence of PPCs first decreased and then increased with increasing NK cell ratio. The proportion of patients with Grade 3 or higher PPCs in the normal group was lower than that in the abnormal group [108 (23.7%) vs. 223 (49%)]. The indwelling time of the thoracic drainage tube in the abnormal group was longer than that in the normal group [3 (3, 4) vs. 3 (3, 5)]. A preoperative abnormal NK cell ratio constituted a risk factor for PPCs in each subgroup. ConclusionLung cancer patients with an abnormal proportion of peripheral blood NK cells before surgery were more likely to develop PPCs, their disease degree was more severe, and they had a prolonged duration of chest tube indwelling. Compared with those with abnormally high NK cell ratios, those with abnormally low NK cell ratios had more pronounced PPCs.

Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

The mechanisms of mAb-induced ADCC have been well established. However, the ADCC bioassays used to quantify mAb-induced ADCC require continued development/refinement to properly assess and compare the potency of newly developed therapeutic mAbs and biosimilars to meet regulatory requirements. We used trastuzumab and a lactate dehydrogenase (LDH)-based ADCC bioassay as a model to define critical parameters of the ADCC bioassay, describing how several bioassay parameters, including preparation of effector cells, E/T ratio, target cell selection, bioassay media components, and treatment time can influence the data quality of the ADCC activity. We confirm that a 4 to 24 h recovery cultivation is required to restore peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cell activity toward ADCC when using cryopreserved PBMCs. Furthermore, we delineated the cellular mechanisms underlying the restored ADCC activity following the recovery cultivation. We observed that CD69, an early marker of NK cell activation, was upregulated and a new subset CD56dim/CD16dim population was dramatically increased in the recovered NK cells, which led to an increase in expression and secretion of perforin, granzyme B, and cytokine production. This study provides comprehensive technical insights into ADCC bioassay optimization to inform trastuzumab biosimilar development. The knowledge gained from this study can also be leveraged to guide bioassay development for therapeutic mAbs with ADCC as the primary mechanism of action.

Pharmacokinetics, pharmacodynamics, and toxicity of a PD-1-targeted IL-15 in cynomolgus monkeys.

e14568 Background: Interleukin (IL)-15 is a potent cytokine that enhances the proliferation and effector functions of IL-2Rβ/γ-expressing lymphocytes, such as NK cells and T cell subtypes (CD8+, CD4+, gd, and NKT). PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. PD-1 is expressed on tumor-infiltrating T cells at relatively high levels. PF-07209960 has reduced activity on PD-1-negative NK cells and enhanced activity on PD-1-expressing intratumoral CD8+ T cells, therefore is expected to have improved therapeutic window. Methods: The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a two-dose PKPD (0.01 – 0.3 mg/kg/dose) study and in a three-dose (SC route) GLP toxicity study (0.1 – 3 mg/kg/dose). Results: PF-07209960 showed dose-dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. The expansion of T cells and NK cells in peripheral blood peaked around Day 8 and waned to near baseline levels by Day 15. Release of cytokines IL-6, IFNɣ, and IL-10 were detected, which peaked at 72 hours postdose. All animals at 3 mg/kg/dose were euthanized in a moribund condition on Day 4 of the dosing phase, coincident with the peak of cytokine release. Four out of six monkeys administered 1 mg/kg were euthanized between Day 6 and Day 16 due to thrombocytopenia, bleeding, and poor physical condition. Microscopic evaluations revealed generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 µg/mL and 37.9 µg*h/mL, respectively. Conclusions: PF-07209960 demonstrated dose-dependent exposure and robust PD (T cell and NK cell subsets expansion and cytokine release) in cynomolgus monkeys, with well-characterized safety profile. In summary, the results of the present study support Phase I clinical evaluation of PF-07209960 in cancer patients.

A phase II trial of monalizumab in combination with durvalumab (MEDI4736) plus platinum-based chemotherapy for first-line treatment of extensive stage small cell lung cancer (MOZART): Hoosier Cancer Research Network LUN21-530 study.

TPS8129 Background: Chemoimmunotherapy consisting of platinum, etoposide, and PD-L1 inhibitor remains the standard of care first-line treatment for extensive stage small cell lung cancer (SCLC) yielding median overall survival (OS) of slightly over a year. NKG2A/CD94 is an immune checkpoint that is selectively expressed on natural killer (NK) cells and CD8+ T cells in the tumor microenvironment. When engaging with its ligand, HLA-E, NKG2A transduces inhibitory signals that suppress immune-mediated cytotoxicity. Thus, NKG2A blockade enhances anti-tumor immunity by promoting both, NK and CD8+ T cell, effector functions. Monalizumab, a monoclonal antibody targeting NKG2A, enhances NK cell activity against tumor cells and rescues CD8+ T cell function in combination with PD-(L)1 axis blockade. Pre-clinical data have demonstrated that the absence of NK cells substantially enhances metastatic dissemination of SCLC tumor cells in vivo. NK cell function is modulated in response to cytotoxic chemotherapy, especially platinum. Post-chemotherapy NK cells display an induced expression of NKG2A compared with pre-chemotherapy patients and is associated with a reduced NK cell mediated anti-tumor activity. Hyperactivation of NK cell activity ameliorates SCLC metastases, an effect that is enhanced when combined with anti PD-(L)1 therapy. Altogether, these data suggest that addition of monalizumab to standard of care first-line treatment may be associated with improved efficacy in patients with SCLC. Methods: Patients with extensive-stage SCLC with ECOG performance status of 0-2 and adequate organ function are eligible for enrollment on this single-arm phase II study with an initial safety lead-in cohort. Patients may have received one prior cycle of platinum doublet with or without durvalumab and may have treated or untreated asymptomatic brain metastasis. Patients will receive platinum (cis or carbo), etoposide, durvalumab, and monalizumab every 3 weeks for 4 cycles followed by durvalumab and monalizumab every 4 weeks until disease progression or unacceptable toxicity. Primary endpoints include 1-year progression-free survival, safety, and tolerability. Secondary endpoints include objective response rate, 1-year overall survival, and intracranial progression-free survival. Exploratory objectives include analyzing association of treatment efficacy with minimal residual disease status, blood- and tissue-based genomic and transcriptomic signatures, tumor infiltrating immune cells, and peripheral blood NK cell and CD8+ T cell activity. Five of the planned 30 patients have been enrolled to date. Clinical trial information: NCT05903092 .

A phase 1 study of REGN6569, a GITR mAb, in combination with cemiplimab in patients (pts) with advanced solid tumor malignancies: Initial dose-escalation results.

2650 Background: REGN6569 is a fully human immunoglobulin G1 monoclonal antibody (mAb) that is highly specific for glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR). GITR is expressed on several immune cell subtypes, notably regulatory T cells (Tregs), and activated natural killer (NK) cells. REGN6569 demonstrated greater in vitro antibody-dependent cell-mediated cytotoxicity against GITR-expressing Tregs, as compared to GITR-expressing CD8+ T cells. Mouse studies showed that REGN6569 + cemiplimab (anti-PD-1) combination treatment achieved longer-term tumor responses compared with either drug alone. Methods: This is a first-in-human study (NCT04465487) evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of REGN6569 administered intravenously (IV) every 3 weeks (Q3W) + cemiplimab 350 mg IV Q3W, in pts with advanced solid tumors for which immune checkpoint inhibitor therapies have not been approved or are not available. The study includes a dose-escalation part with 5 dose levels (DL1-DL5) for REGN6569 (“4+3” design), with an initial dose of REGN6569 monotherapy given as a safety lead-in followed by REGN6569 + cemiplimab in subsequent doses. Results: As of the data cutoff date Oct 20, 2023, the dose-escalation part has been completed. 29 pts (median age 58.0 years, 62.1% male) were treated with REGN6569 + cemiplimab, up to the 1200 mg dose level (DL5), across many solid tumor types. The most common tumor type was colorectal cancer (34.5%). One pt (40 mg DL2) experienced a dose-limiting toxicity (Grade 3 hepatic failure); maximum tolerated dose was not reached. Twelve pts (41.4%) had a Grade ≥3 treatment-emergent adverse event (TEAE) and 16 pts (55.2%) had a treatment-related TEAE (any grade). The most frequent TEAEs (any grade) were arthralgia (24.1%), infusion-related reactions, and abdominal pain (20.7% each). There were no treatment-related deaths; 19 (65.5%) pts had disease progression leading to death. Two pts achieved ongoing partial responses by investigator assessment with REGN6569 + cemiplimab treatment: 1 pt with mucoepidermoid tumor of the parotid gland treated with 120 mg (DL3) REGN6569 and 1 pt with B3 thymoma treated with 400 mg (DL4) REGN6569, with duration of responses, 5.6 and 10.4 months, respectively. Full receptor occupancy on circulating Tregs was observed in all dose cohorts following REGN6569 treatment. Increased frequency (~10–50%) of proliferating NK cells in peripheral blood was observed post REGN6569 treatment across all dose cohorts. Conclusions: In this dose-escalation study, REGN6569 was administered up to 1200 mg (DL5) in combination with cemiplimab with one dose-limiting toxicity. The study has progressed to dose-expansion cohorts in anti–PD-1-resistant head and neck cancer, with pts treated with REGN6569 (DL5) + cemiplimab. Clinical trial information: NCT04465487 .