Receptors NKp46 Research Articles

Biopsy-free profiling of the uterine immune system in patients with recurrent pregnancy loss and unexplained infertility

Research questionWhat are the differences in menstrual blood lymphocytes between controls, patients with recurrent pregnancy loss (RPL) and patients with unexplained infertility (uINF)? DesignProspective study including 46 healthy controls, 28 RPL and 11 uINF patients. A feasibility study compared lymphocyte compositions of endometrial biopsies and menstrual blood collected during the first 48 h of menstruation in seven controls. In all patients, peripheral and menstrual blood from the first and subsequent 24 h were analysed separately by flow cytometry, focusing on the main lymphocyte populations and natural killer (NK) cell subsets. ResultsThe first 24 h of menstrual blood resembles the uterine immune milieu as tested by endometrial biopsy. RPL patients showed significantly higher menstrual blood CD56+ NK cell numbers than controls (mean ± SD: 31.13 ± 7.52% versus 36.73 ± 5.4%, P = 0.002). Menstrual blood CD56dimCD16bright NK cells within the CD56+ NK cell population were decreased in RPL (16.34 ± 14.65%, P = 0.011) and uINF (15.7 ± 5.91%, P = 0.02) patients versus control (20.42 ± 11.53%). uINF patients had the lowest menstrual blood CD3+ T cell counts (38.81 ± 5.04%, control versus uINF: P = 0.01) and cytotoxicity receptors NKp46 and NKG2D on CD56brightCD16dim cells were higher in uINF (68.12 ± 11.84%, P = 0.006; 45.99 ± 13.83%, P = 0.01, respectively) and RPL (NKp46: 66.21 ± 15.36%, P = 0.009) patients versus controls. RPL and uINF patients had higher peripheral CD56+ NK cell counts versus controls (11.42 ± 4.05%, P = 0.021; 12.86 ± 4.29%, P = 0.009 versus 8.4 ± 3.5%). ConclusionsCompared with controls, RPL and uINF patients had a different menstrual blood-NK-subtype profile, indicating an altered cytotoxicity. In future studies, this non-invasive analysis might enable identification and monitoring of patients receiving immunomodulatory medications.

Differential Expression of LLT1, SLAM Receptors CS1 and 2B4 and NCR Receptors NKp46 and NKp30 in Pediatric Acute Lymphoblastic Leukemia (ALL).

Acute lymphoblastic leukemia (ALL) represents the most common pediatric cancer. Most patients (85%) develop B-cell ALL; however, T-cell ALL tends to be more aggressive. We have previously identified 2B4 (SLAMF4), CS1 (SLAMF7) and LLT1 (CLEC2D) that can activate or inhibit NK cells upon the interaction with their ligands. In this study, the expression of 2B4, CS1, LLT1, NKp30 and NKp46 was determined. The expression profiles of these immune receptors were analyzed in the peripheral blood mononuclear cells of B-ALL and T-ALL subjects by single-cell RNA sequencing data obtained from the St. Jude PeCan data portal that showed increased expression of LLT1 in B-ALL and T-ALL subjects. Whole blood was collected from 42 pediatric ALL subjects at diagnosis and post-induction chemotherapy and 20 healthy subjects, and expression was determined at the mRNA and cell surface protein level. A significant increase in cell surface LLT1 expression in T cells, monocytes and NK cells was observed. Increased expression of CS1 and NKp46 was observed on monocytes of ALL subjects at diagnosis. A decrease of LLT1, 2B4, CS1 and NKp46 on T cells of ALL subjects was also observed post-induction chemotherapy. Furthermore, mRNA data showed altered expression of receptors in ALL subjects pre- and post-induction chemotherapy treatment. The results indicate that the differential expression of the receptors/ligand may play a role in the T-cell- and NK-cell-mediated immune surveillance of pediatric ALL.

Alterations in the immune system persist after one year of convalescence in severe COVID-19 patients.

Severe COVID-19 originates a myriad of alterations in the immune system during active disease, especially in the T and NK cell compartments, but several studies in the last year have unveiled some alterations that persist in convalescence. Although most of the studies follow the participants for a short recovery time, studies following patients up to three or six months still find alterations. We aimed at evaluating changes in the NK, T and B cell compartments after severe COVID-19 in participants with a median recovery time of eleven months. Eighteen convalescent of severe COVID-19 (CSC), 14 convalescent of mild COVID-19 (CMC) and nine controls were recruited. NKG2A, NKG2C, NKG2D and the activating receptor NKp44 were evaluated in NKbright, NKdim and NKT subpopulations. In addition, CD3 and CD19 were measured and a basic biochemistry with IL-6 levels was obtained. CSC participants showed lower NKbright/NKdim ratio, higher NKp44 expression in NKbright subpopulations, higher levels of serum IL-6, lower levels of NKG2A+ T lymphocytes and a trend to a lower expression of CD19 in B lymphocytes compared to controls. CMC participants showed no significant alterations in the immune system compared to controls. These results are concordant with previous studies, which find alterations in CSC weeks or months after resolution of the symptoms, and point to the possibility of these alterations lasting one year or more after COVID-19 resolution.

Affinity Maturation of the Natural Ligand (B7-H6) for Natural Cytotoxicity Receptor NKp30 by Yeast Surface Display.

In recent years, the development of bispecific antibodies (bsAbs) has experienced tremendous progress for disease treatment, and consequently, a plethora of bsAbs is currently scrutinized in clinical trials. Besides antibody scaffolds, multifunctional molecules referred to as immunoligands have been developed. These molecules typically harbor a natural ligand entity for the engagement of a specific receptor, while binding to the additional antigen is facilitated by an antibody-derived paratope. Immunoligands can be exploited to conditionally activate immune cells, e.g., natural killer (NK) cells, in the presence of tumor cells, ultimately causing target-dependent tumor cell lysis. However, many ligands naturally show only moderate affinities toward their cognate receptor, potentially hampering killing capacities of immunoligands. Herein, we provide protocols for yeast surface display-based affinity maturation of B7-H6, the natural ligand of NK cell-activating receptor NKp30.

Non-fitness status of peripheral NK cells defined by decreased NKp30 and perforin, and increased soluble B7H6, in cervical cancer patients.

The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30+ NK cells, especially in the CD56dim subpopulation, was found. Perforin levels were decreased in these cells. Decreased expression of the NKp30 C isoform and overexpression of soluble B7H6 was found in cervical cancer patients when compared against healthy subjects. PBMCs from healthy subjects downregulated NKp30 isoforms after co-culture with B7H6-expressing tumour cells. Taken together, these findings describe a unique down-modulation or non-fitness status of the immune response in cervical cancer, the understanding of which will be important for the design of novel immunotherapies against this disease.

MiR-221-5p and miR-186-5p Are the Critical Bladder Cancer Derived Exosomal miRNAs in Natural Killer Cell Dysfunction

Bladder cancer (BC) is the tenth most commonly diagnosed cancer worldwide, and its carcinogenesis mechanism has not been fully elucidated. BC is able to induce natural killer (NK) cell dysfunction and escape immune surveillance. The present study found that exosomes derived from the urinary bladder cancer cell line (T24 cell) contribute in generating NK cell dysfunction by impairing viability, and inhibiting the cytotoxicity of the NK cell on target cells. Meanwhile, T24 cell-derived exosomes inhibited the expression of the important functional receptors NKG2D, NKp30, and CD226 on NK cells as well as the secretion of perforin and granzyme-B. The critical miRNAs with high expression in T24 cell-derived exosomes were identified using high-throughput sequencing. Furthermore, following dual-luciferase reporter assay and transfection experiments, miR-221-5p and miR-186-5p were confirmed as interfering with the stability of the mRNAs of DAP10, CD96, and the perforin gene in NK cells and may be potential targets used in the therapy for BC.

Functional molecular expression of nature killer cells correlated to HBsAg clearance in HBeAg-positive chronic hepatitis B patients during PEG-IFN α-2a therapy.

To explore whether the frequencies and functional molecules expression of Natural Killer cells (NK cells) are related to hepatitis B surface antigen (HBsAg) disappearance in hepatitis B e envelope antigen (HBeAg)-positive patients with chronic hepatitis B (CHB) throughout peginterferon alpha-2a (PEG-IFN α-2a) treatment. In this prospective research, HBeAg-positive patients with CHB received PEG-IFN α-2a treatment, completing 4-year follow-up. After PEG-IFN α-2a treatment, undetectable HBV DNA, HBsAg loss, and HBeAg disappearance were defined as functional cure. Proportions of NK, CD56dim, CD56bright, NKp46+, NKp46dim, NKp46high, and interferon alpha receptor 2 (IFNAR2)+ NK cells, and the mean fluorescence intensity (MFI) of NK cell surface receptors IFNAR2 and NKp46 were detected. 66 patients were enrolled into the study in which 17 patients obtained functional cure. At baseline, hepatitis B virus desoxyribose nucleic acid (HBV DNA) titer in patients with functional cure was remarkably lower than that in Non-functional cure group. Compared with baseline, HBV DNA levels, HBsAg levels, and HBeAg levels significantly declined at week 12 and 24 of therapy in patients with functional cure. At baseline, the negative correlation between CD56bright NK% and HBV DNA and the negative correlation between CD56dim NK% and HBV DNA was showed; CD56bright NK% and IFNAR2 MFI in patients with functional cure were remarkably higher than those in patients without functional cure. After therapy, CD56bright NK% and NKp46high NK% in patients with functional cure were higher than those in patients without functional cure. In Functional cure group, after 24 weeks of treatment NK%, CD56bright NK%, IFNAR2 MFI weakly increased, and NKp46high NK% and NKp46 MFI significantly increased, meanwhile, CD56dim NK% and NKp46dim NK% decreased. Only NKp46 MFI increased after therapy in patients without functional cure. The lower HBV DNA load and the higher CD56bright NK% before therapy, and the higher the post-treatment CD56bright NK%, IFNAR2 MFI, NKp46high NK%, the easier to achieve functional cure.

IPH6501 Is a Novel NKp46-Targeting Tetraspecific Antibody-Based Natural Killer Cell Engager Therapeutic (ANKET) Armed with a Non-Alpha IL-2 Variant and Developed for the Treatment of CD20-Positive Malignancies

CD20 is expressed by >90% of B-cell non-Hodgkin's lymphomas (NHL). Several generations of CD20-targeting monoclonal antibodies including rituximab, ofatumumab, and obinutuzumab have been widely used for B-cell malignancy therapies. Despite the recent approvals of novel CD20-targeting agents, new alternatives and strategies are still required for patients, which are relapsing or refractory after several lines of treatment. High circulating NK cell numbers have been associated with better clinical responses to anti-CD20- targeting monoclonal antibodies, supporting the role of NK cells in efficacy of these treatments. IPH6501, a novel antibody-based NK cell engager therapeutics (ANKET), is a single tetraspecific molecule engaging two NK cell activating receptors NKp46 and CD16a (FcγRIIIa), the β chain (CD122) of the interleukin-2 receptor (IL-2R), and the CD20 antigen expressed on malignant B cells. The IL-2R binding element incorporated in IPH6501 is an IL-2 variant (IL-2v) designed with point mutations that abolish binding to the IL-2R-α chain (CD25), with the goal of limiting toxicity and interaction with Tregs. IL-2v incorporated into IPH6501 is directed towards NK cells through the binding with high affinity to NKp46 and CD16a, providing its ability to interact with IL-2R preferentially on NK cells and to promote their activation and proliferation at pM doses. IPH6501 was designed to induce NK cell mediated-cytotoxicity and cytokine secretion by co-engaging CD16a and NKp46. Only the binding of IPH6501 to CD20, bridging the NK cells to the target cells, was able to trigger the cytotoxic activity of NK cells. Non-saturating doses of IPH6501 on NK cells and on CD20+ cells were sufficient to promote maximal killing activity. Furthermore, IPH6501 promoted NK cell cytotoxicity against tumor cells expressing very low levels of CD20. IPH6501 also demonstrated superiority to control tumor cell growth in vitro, as compared to the CD20-targeting clinical benchmark antibodies rituximab and the Fc-optimized obinutuzumab. In vivo treatment with a mouse surrogate of IPH6501 induced peripheral NK cell proliferation and activation, and demonstrated potent antitumor efficacy in a xenograft mouse model using the aggressive human B-lymphoma CD20+ RAJI cell line engrafted subcutaneously. In non-human primate, a well-tolerated dose of IPH6501 led to peripheral NK cell expansion and to the depletion of CD20+ B cells in the circulation and within lymphoid tissues, with minimal systemic cytokine release. In conclusion, IPH6501 is a first-in-class CD20-targeting tetraspecific NK cell engager designed to promote NK cell proliferation and specific cytotoxicity against CD20+ cells. IPH6501 is thus a promising biologic designed to harness the anti-tumor functions of NK cells in CD20+ B cell malignancies. IND-enabling studies are ongoing, and IPH6501 is expected to enter FIH clinical study in 2023.

SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity.

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

CD56bright CD16- natural killer cells as an important regulatory mechanism in chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity after hematopoietic stem cell transplantation (HSCT). In large patient populations, we have shown a CD56bright natural killer (NK) population to strongly associate with a lack of cGvHD and we hypothesize that these cells function to suppress cGvHD. We aimed to isolate and define the characteristics of regulatory NK (NKreg) cells associated with suppression of cGvHD. Immunophenotypic evaluation of a large pediatric population found the CD56bright NK population associated with a lack of cGvHD to be perforin-, Granzyme B-, and CD335+. Transcriptome analysis of a small patient cohort of CD56bright compared to CD56dim NK cells found the NKreg cells to also overexpress Granzyme K, IL-7R, GPR183, RANK, GM-CSFR, TCF7, and IL23A. Further analysis of this CD56bright NKreg population found a subpopulation that overexpressed IRF1, and TNF. We also found that viable NKreg cells may be isolated by sorting on CD56+ and CD16- NK cells, and this population can suppress allogeneic CD4+ T cells, but not Treg cells or CD8+ T cells through a non-cytolytic, cell-cell contact dependent mechanism. Suppression was not reliant upon the NKp44, NKp46, or GPR183 receptors. Additionally, NKreg cells do not kill leukemic cells. Moreover, this is the first paper to clearly establish that a CD56brightCD3-CD16-perforin- NKreg population associates with a lack of cGvHD and has several unique characteristics, including the suppression of helper T-cell function in vitro. With further investigation we may decipher the mechanism of NKreg suppression and operationalize expansion of NKreg cells associated with cGvHD suppression.

Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant

SummaryHarnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the β-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20+ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.

Liver mesenchymal stem cells are superior inhibitors of NK cell functions through differences in their secretome compared to other mesenchymal stem cells.

Liver-resident mesenchymal stem cells (L-MSCs) are superior inhibitors of alloreactive T cell responses compared to their counterparts from bone marrow (BM-MSCs) or adipose tissue (A-MSCs), suggesting a role in liver’s overall tolerogenic microenvironment. Whether L-MSCs also impact NK cell functions differently than other MSCs is not known. We generated and characterized L-MSCs, A-MSCs and BM-MSCs from human tissues. The mass spectrometry analysis demonstrated that L-MSC secretome is uniquely different than that of A-MSC/BM-MSC, with enriched protein sets involved in IFNγ responses and signaling. When co-cultured with primary human NK cells, L-MSCs but not other MSCs, decreased surface expression of activating receptors NKp44 and NKG2D. L-MSCs also decreased IFNγ secretion by IL-2-stimulated NK cells more effectively than other MSCs. Cytolytic function of NK cells were reduced significantly when co-cultured with L-MSCs, whereas A-MSCs or BM-MSCs did not have a major impact. Mechanistic studies showed that the L-MSC-mediated reduction in NK cell cytotoxicity is not through changes in secretion of the cytotoxic proteins Perforin, Granzyme A or B, but through increased production of HLA-C1 found in L-MSC secretome that inhibits NK cells by stimulating their inhibitory receptor KIRDL2/3. L-MSCs are more potent inhibitors of NK cell functions than A-MSC or BM-MSC. Combined with their T cell inhibitory features, these results suggest L-MSCs contribute to the tolerogenic liver microenvironment and liver-induced systemic tolerance often observed after liver transplantation.

Structural studies offer a framework for understanding the role of properdin in the alternative pathway and beyond.

Structures of alternative pathway proteins have offered a comprehensive structural basis for understanding the molecular mechanisms governing activation and regulation of the amplification pathway of the complement cascade. Although properdin (FP) is required in vivo to sustain a functional alternative pathway, structural studies have been lagging behind due to the extended structure and polydisperse nature of FP. We review recent progress with respect to structure determination of FP and its proconvertase/convertase complexes. These structures identify in detail regions in C3b, factor B and FP involved in their mutual interactions. Structures of FP oligomers obtained by integrative studies have shed light on how FP activity depends on its oligomerization state. The accumulated structural knowledge allows us to rationalize the effect of point mutations causing FP deficiency. The structural basis for FP inhibition by the tick CirpA proteins is reviewed and the potential of alphafold2 predictions for understanding the interaction of FP with other tick proteins and the NKp46 receptor on host immune cells is discussed. The accumulated structural knowledge forms a comprehensive basis for understanding molecular interactions involving FP, pathological conditions arising from low levels of FP, and the molecular strategies used by ticks to suppress the alternative pathway.

OP-24 - Analysis of expression intensity of NK cell receptor in peritoneal fluid in endometriosis

To clarify the details of peritoneal fluid NK (pfNK) cell involvement in endometriosis, we investigated the expression of anctivating and inhibitory receptors on pfNK cells and cytokine production by pfNK cells in endometriosis. The patients who need surgical treatment for Endometriosis (endometriosis group, n=32) and other gynecological benign disease without endometriosis (controls, n=30) were included for this study. Peritoneal fluid were collected at the time of surgery. The exprssion of pfNK cells (CD56+ cells) were assayed for activating receptors (CD16, NKp46, NKG2C, NKG2D), inhibitory receptors (CD158a, NKG2A), and intra-cellular cytokines (TNF-α, IFN-γ, IL-4, IL-10, TGF-β) by 6 flow cytometry. CD56 dim /NKp46 + NK cells were significantly decreased in endometriosis group compared to controls. (p<0.05). Furthermore, NKG2C + /NKp46 + NK cells were significantly decreased in endometriosis group compared to controls. (p<0.05) On the other hand, NKp46 + /NKG2D + NK cells were significant increased in endometriosis group compared to controls (p<0.05). IFN-γ-producing NK cells in endometriosis group was significantly higher than that in controls (P<0.05). pf NK cells differ in cytotoxicity according to the expression intensity of NKG2C, NKG2D, and NKp46 receptors, which may be related to their abnormal cytokine production. In women with endometriosis, the decrease of cytotoxicity for pf NK cells causes the progression of pelvic endometriosis.

756P Glypican-3 (GPC3) and NKp46 directed FLEX-NK engager antibody (CYT-303) recruits natural killer (NK) cells to tumors in a preclinical hepatocellular carcinoma (HCC) mouse model

GPC3 is an oncofetal antigen that is highly expressed in HCC while minimally expressed in adult normal tissues except placenta. CYT-303 is a multifunctional bispecific NK engager targeting NK cell activating receptor NKp46 and GPC3 expressed on tumor cells constructed with Cytovia’s Flex-NK scaffold with a fully functional Fc. Previously, we reported CYT-303 in vitro treatment of Hep3B tumor cells demonstrated cytolysis and cytokine production as well as in vivo tumor growth inhibition when combined with both PBNK and iNK cells. Here we further characterized the CYT-303 dose-response for tumor growth inhibition in vivo and its influence on NK cell distribution. CYT -303 pharmacokinetics was evaluated in tumor-bearing mice. Anti-tumor efficacy and dose response of CYT-303 in the presence of human PBNKs or iNKs was evaluated in NSG-IL15 mice bearing subcutaneous Hep3B tumors. Biodistribution of the NK cells were analyzed by flow cytometry. CYT-303 pharmacokinetics in tumor bearing mice was similar to normal mice suggesting no target mediated clearance. In the Hep3B tumor model in PBNK or iNK injected NSG-hIL-15 mice, CYT-303 showed dose dependent tumor growth inhibition compared to control hIgG1 treated mice. Consistent with CYT-303 tumor growth inhibition, increased NK cells were detected in the tumor compared to control animals. Blood NK cells in CYT-303 treated animals were significantly lower compared to IgG1 isotype control treated mice suggesting CYT-303 could facilitate NK cell trafficking from blood into the tumor. Blood AFP (alpha fetoprotein), a biomarker in HCC, decreased with CYT-303 tumor growth inhibition. Pharmacologically active CYT-303 doses were identified. CYT-303 can help recruit NK cells to the tumor site to achieve tumor growth inhibition.

Abstract PO001: Preclinical activity of glypican-3 (GPC3) and NKp46 directed FLEX-NK™ engager antibody (CYT-303) in combination with iPSC derived natural killer cells (iNKs) or peripheral blood (PB) NK cells in hepatocellular carcinoma (HCC)

Abstract BACKGROUND: GPC3 is highly expressed in HCC while it is hardly expressed in adult normal tissues except placenta. Given the presence of dysfunctional NK cells in the HCC tumor microenvironment that contribute to poor anti-tumor immune responses, we sought to develop an iNK cell therapy in combination with GPC3 targeted NK cell engagers (NKE). FLEX-NK™ is a proprietary tetravalent IgG1-like multifunctional antibody NKE platform with a novel FLEX-linker to allow for simultaneous binding to different antigens on different cells. CYT-303 was constructed to engage NK cells via the activation receptor NKp46 and to target cancer cells via GPC3. We hypothesized that CYT-303 may be active in the tumor microenvironment where there are sufficient number and function of endogenous NK cells, but that this activity may be augmented by fresh functional NK cells. iNK cells derived from iPSC, a uniform starting material with unlimited self-renewal capabilities, can be expanded to produce a universal off-the-shelf allogeneic therapy. We investigated the combination of CYT-303 to enhance tumor specificity and potency of iNKs. METHODS: CYT-303 was expressed in CHOZEN cells and purified by step column chromatography. iNK’s were differentiated from iPSC’s and expanded using irradiated feeder cells. PB-NK cells were purified from healthy donor blood. iNK and PB-NK cytotoxicity against Hep3B tumors was evaluated in the presence of CYT-303 or isotype control antibodies at a fixed E/T ratio. CYT-303 pharmacokinetics in mice was evaluated following a single intravenous injection. Anti-tumor efficacy of the iNK combination with CYT-303 was evaluated in NSG-IL15 mice bearing subcutaneous Hep3B tumors following intra-tumoral injection of iNKs and intravenous dosing with CYT-303. Blood alpha-fetoprotein (AFP) levels were assessed by immunoassay. Cytokine release assessments for CYT-303 was evaluated in an in-vitro PBMC activation assay. RESULTS: iNK cells express high levels of multiple activation receptors including NKp46, but low levels of CD16. Despite low expression of CD16, natural cytotoxicity of iNK’s against Hep3B tumors was higher than with fresh PB-NK and CYT-303 redirected killing of Hep3B tumors was comparable to PB-NK’s. The CYT-303 pharmacokinetics study in mice showed a terminal half-life of 17 hrs. In a Hep3B tumor model in NSG-hIL-15 mice, the combination of CYT-303 and iNK’s showed significantly greater tumor growth inhibition compared to iNK’s alone plus an IgG1 isotype control. Blood AFP levels decreased in the CYT-303 plus iNK combination compared to iNK’s alone. Cytokine release assessment of CYT-303 in the human PBMC assay showed no evidence of cytokine release while high levels of cytokine release was observed with anti-CD28 (TGN1412) and CD3 antibody controls. CONCLUSIONS: The combination of NK cell engager CYT-303 and iNK cells is active in preclinical models of HCC with low levels of cytokine release. These data support further development of CYT-303 and iNKs to engage and empower NK cells as an off the shelf therapeutic approach in HCC. Citation Format: Antonio Arulanandam, Liang Lin, Hao-Ming Chang, Harish Potu, Vishal Khairnar, David Zou, Melissa Triggiano, Nejmi Dilmac, Yinan Yang, Shira Kahlon, Stanley Frankel, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Yoav Hershkovitz, Yaron Ilan, Wei Li. Preclinical activity of glypican-3 (GPC3) and NKp46 directed FLEX-NK™ engager antibody (CYT-303) in combination with iPSC derived natural killer cells (iNKs) or peripheral blood (PB) NK cells in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO001.

Decreased CD57 expression of natural killer cells enhanced cytotoxicity in patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory biliary disease for which the immunopathological basis remains an enigma. Natural killer (NK) cells are key components of innate immunity and seemingly play diversified roles in different autoimmune disorders (AIDs). The aim of this study was to determine the role of NK cells in the pathogenesis of PSC. The frequency and phenotype of circulating NK cells in a large cohort of patients with PSC and healthy controls (HCs) were systematically examined. In addition, the functional capacity of NK cells including cytotoxicity and cytokine production was studied. The frequency of CD3-CD56dimCD16+ (defined as CD56dim) NK cells in PSC patients was significantly lower in comparison to HCs. CD56dim NK cells from PSC displayed a more immature phenotype including high expression of the natural killing receptor NKp46 and downregulation of the highly differentiated NK cell marker CD57. Interestingly, the reduction of CD57 expression of NK cells was associated with the disease severity of PSC. In addition, PSC CD56dim NK cells exhibited increased CD107a degranulation and cytolytic activity toward target cells compared with HCs. Further analysis demonstrated that CD57-CD56dim NK cells from PSC had elevated expression of NKp46, NKp30, IL-2 receptor, and KLRG1 and higher cytotoxic capacity as compared to CD57+CD56dim NK cells. Our data demonstrate that the differentiation of PSC NK cells is dysregulated with enhanced cytotoxic activity. This change is likely to be functionally involved in pathogenesis and disease progression, deducing the potential of NK-directed immunotherapy for PSC.

Self-carried nanodrug (SCND-SIS3): A targeted therapy for lung cancer with superior biocompatibility and immune boosting effects

Transforming growth factor β (TGF-β) is a well-known key mediator for the progression and metastasis of lung carcinoma. However, cost-effective anti-TGF-β therapeutics for lung cancer remain to be explored. Specifically, the low efficacy in drug delivery greatly limits the clinical application of small molecular inhibitors of TGF-β. In the present study, specific inhibitor of Smad3 (SIS3) is developed into a self-carried nanodrug (SCND-SIS3) using the reprecipitation method, which largely improves its solubility and bioavailability while reduces its nephrotoxicity. Compared to unmodified-SIS3, SCND-SIS3 demonstrates better anti-cancer effects through inducing tumor cell apoptosis, inhibiting angiogenesis, and boosting NK cell-mediated immune responses in syngeneic Lewis Lung Cancer (LLC) mouse model. Better still, it could achieve comparable anti-cancer effect with just one-fifth the dose of unmodified-SIS3. Mechanistically, RNA-sequencing analysis and cytokine array results unveil a TGF-β/Smad3-dependent immunoregulatory landscape in NK cells. In particular, SCND-SIS3 promotes NK cell cytotoxicity by ameliorating Smad3-mediated transcriptional inhibition of Ndrg1. Furthermore, improved NK cell cytotoxicity by SCND-SIS3 is associated with higher expression of activation receptor Nkp46, and suppressed levels of Trib3 and TSP1 as compared with unmodified-SIS3. Taken together, SCND-SIS3 possesses superior anti-cancer effects with enhanced bioavailability and biocompatibility, therefore representing as a novel therapeutic strategy for lung carcinoma with promising clinical potential.

Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion.

NK cell exhaustion is caused by chronic exposure to activating stimuli during viral infection, tumorigenesis, and prolonged cytokine treatment. Evidence suggests that exhaustion may play a role in disease progression. However, relative to T cell exhaustion, the mechanisms underlying NK cell exhaustion and methods of reversing it are poorly understood. Here, we describe a potentially novel in vitro model of exhaustion that uses plate-bound agonists of the NK cell activating receptors NKp46 and NKG2D to induce canonical exhaustion phenotypes. In this model, prolonged activation resulted in downregulation of activating receptors, upregulation of checkpoint markers, decreased cytokine production and cytotoxicity in vitro, weakened glycolytic capacity, and decreased persistence, function, and tumor control in vivo. Furthermore, we discovered a beneficial effect of NK cell inhibitory receptor signaling during exhaustion. By simultaneously engaging the inhibitory receptor NKG2A during activation in our model, cytokine production and cytotoxicity defects were mitigated, suggesting that balancing positive and negative signals integrated by effector NK cells can be beneficial for antitumor immunity. Together, these data uncover some of the mechanisms underlying NK cell exhaustion in humans and establish our in vitro model as a valuable tool for studying the processes regulating exhaustion.

Abstract 3436: Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells

Abstract Given that CD38 is a clinically validated target for NK cell mediated cytotoxicity in multiple myeloma, we sought to leverage our FLEX NKTrademark platform to create a NK engager antibody targeting CD38. FLEX NKTrademark is a proprietary platform for production of tetravalent multifunctional NK engager antibodies with a novel FLEX linker to allow for simultaneous binding of both the targeted cancer cells and NK cells. NK engagement and activation is mediated through a binder directed against the natural cytotoxicity receptor NKp46. With the FLEX NKTrademark scaffold, we created two novel tetravalent NK cell engagers targeting CD38, one with a wild type (WT) Fc and one with a mutant null Fc. Both CD38 NKp46 engagers showed dose dependent binding to CD38 expressing multiple myeloma cell lines MM1S and KMS11 and no binding to a CD38 knock out MM.1S cell line. Both engagers also bound multiple myeloma cell lines with ~ 2-fold higher mean fluorescence intensity than anti-CD38 monoclonal antibody (mAb)or daratumumab alone. Epitope mapping studies for our anti-CD38 mAb using alanine scanning mutagenesis showed that amino acid S274 on CD38, critical for binding to daratumumab, is not important for our anti-CD38 antibody binding to CD38, suggesting a distinct epitope detected by our antibody. Interestingly, the NKp46 monoclonal antibody alone without a functional Fc induced peripheral blood NK cell cytolysis of the multiple myeloma cells, consistent with a prior report that NKp46 plays a key role in NK-cell mediated killing of myeloma cells. Both CD38 NKp46 engagers showed further enhanced dose dependent NK cell redirected cytolysis and degranulation against multiple myeloma cells compared to anti-NKp46 monoclonal antibody. The CD38 NKp46 engagers demonstrated higher cytotoxicity than the anti-CD38 binder mAb or daratumumab alone. The CD38 NKp46 engager with the WT Fc is more potent in induction of TNF-α and IFN-ɣ production compared to daratumumab and the engager with the mutant Fc. No IL-1β or IL-6 was induced by the engagers or daratumumab. Daratumumab treatment resulted in NK cell fratricide, while no fratricide was observed with the CD38-NKp46 NK engager with the mutant Fc. In peripheral blood mononuclear cell hemato-toxicity studies depletion of monocytes and NK cells were observed with daratumumab but no depletion was observed with the CD38 NKp46 engager with the mutant Fc. These results suggest that the CD38 NKp46 engagers have a favorable NK cell engager profile for targeting CD38 expressing multiple myeloma that’s distinct from daratumumab. Citation Format: Liang Lin, Hao-Ming Chang, Cecilia Nakid, Stanley Frankel, Dennis Wu, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Jean-Christophe Bories, Antonio Arulanandam, Wei Li. Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3436.