Specific Cytolytic Activity Research Articles

Cytolysin A is an intracellularly induced and secreted cytotoxin of typhoidal Salmonella

Typhoidal Salmonella enterica serovars, such as Typhi and Paratyphi A, cause severe systemic infections, thereby posing a significant threat as human-adapted pathogens. This study focuses on cytolysin A (ClyA), a virulence factor essential for bacterial dissemination within the human body. We show that ClyA is exclusively expressed by intracellular S. Paratyphi A within the Salmonella-containing vacuole (SCV), regulated by the PhoP/Q system and SlyA. ClyA localizes in the bacterial periplasm, suggesting potential secretion. Deletion of TtsA, an essential Type 10 Secretion System component, completely abolishes intracellular ClyA detection and its presence in host cell supernatants. Host cells infected with wild-type S. Paratyphi A contain substantial ClyA, with supernatants capable of lysing neighboring cells. Notably, ClyA selectively lyses macrophages and erythrocytes while sparing epithelial cells. These findings identify ClyA as an intracellularly induced cytolysin, dependent on the SCV environment and secreted via a Type 10 Secretion System, with specific cytolytic activity.

Spinster Homologue 2 Expression Correlates With Improved Patient Survival in Hepatocellular Carcinoma Despite Association With Lymph-Angiogenesis.

Spinster homologue 2 (SPNS2) is a transporter of sphingosine-1-phosphate (S1P), a bioactive lipid linked to cancer progression. We studied the link between SPNS2 gene expression, tumor aggressiveness, and outcomes in patients with hepatocellular carcinoma (HCC). Gene expression in patients with HCC was analyzed from the Cancer Genome Atlas (TCGA) (n = 350) and GSE76427 (n = 115) as a validation cohort, as well as liver tissue cohort GSE6764 (n = 75). High-SPNS2 HCC was significantly associated with high level of lymph-angiogenesis-related factors. SPNS2 expression was significantly higher in normal liver and early HCC versus advanced HCC (P < 0.02). High SPNS2 levels enriched immune response-related gene sets; inflammatory, interferon (IFN)-α, IFN-γ responses, and tumor necrosis factor (TNF)-α, interleukin (IL)-6/Janus kinase/signal transducer and activator of transcription (JAK/STAT3) signaling, complement and allograft rejection, but did not significantly infiltrate specific immune cells nor cytolytic activity score. High-SPNS2 HCC enriched tumor aggravating pathway gene sets such as KRAS (Kirsten rat sarcoma virus) signaling, but inversely correlated with Nottingham histological grade, MKI67 (marker of proliferation Ki-67) expression, and cell proliferation-related gene sets. Further, high-SPNS2 HCC had significantly high infiltration of stromal cells, showing that low-SPNS2 HCC is highly proliferative. Finally, high-SPNS2 HCC was associated with better disease-free, disease-specific, and overall survival (P = 0.031, 0.046, and 0.040, respectively). Although SPNS2 expression correlated with lymph-angiogenesis and other cancer-promoting pathways, it also enriched immune response. SPNS2 levels were higher in normal liver compared to HCC, and inversely correlated with cancer cell proliferation and better survival. SPNS2 expression may be beneficial in HCC patients despite detrimental in-vitro effects.

503 Engineering GBM-specific T Cells Using Photothermal Prussian Blue Nanoparticles

INTRODUCTION: Adoptive T cell therapy (ATCT) has successfully treated hematological malignancies and is currently under investigation for solid tumor therapy. In contrast to existing chimeric antigen receptor (CAR) T cell and/or antigen-specific T cell approaches, which target defined antigens, our PBNP-based approach can target a broad repertoire of antigens in GBM in an antigen-agnostic manner. METHODS: We treated GBM cells with PBNP-based photothermal therapy (PBNP-PTT) prior to culturing with dendritic cells (DCs), and subsequent stimulation of T cells. RESULTS: When PBNP-PTT was administered to U87 cells at an immunogenic thermal dose, we effectively expanded U87-specific T cells. DCs cultured ex vivo with PBNP-PTT-treated U87 cells enabled 15- 30-fold expansion of CD4+ and CD8+ T cells. Upon co-culture with target U87 cells, these T cells secreted IFN-G in a specific and dose-dependent manner (up to 647-fold over controls). T cells manufactured using PBNP-PTT elicited specific cytolytic activity against target U87 cells (33-93% killing at an E:T ratio of 20:1), while sparing normal human astrocytes. Similarly, PBNP-PTT on SNB19 cells resulted in a 7-39-fold expansion of T cells, which elicited 25-66% killing of SNB19 cells at an E:T ratio of 20:1. U87-luc cells were then orthotopically inoculated into NSG mice to establish xenograft GBM, and U87-specific T cells developed via PBNP-PTT were intracranially injected. These T cells eliminated U87 tumors in vivo and significantly improved mouse survival compared to mice treated with PBS (p = 0.0047) or non-specific T cells (p = 0.0123) from the same donor. CONCLUSIONS: These findings provide proof-of-concept data supporting the use of PBNP-PTT to stimulate and expand tumor-specific T cells ex vivo for potential use as an ATCT approach for patients with GBM. We are currently using patient-derived tumor samples to generate IND-enabling data.

CD276 (B7-H3) Is an Immunotherapeutic Target in Acute Myeloid Leukemia with Preclinical Efficacy of Vobramitamab Duocarmazine, an Investigational CD276 Antibody-Drug Conjugate

CD276 (B7-H3) is an immune checkpoint inhibitor expressed in a majority of solid tumors and hematologic malignancies, however it has not been well-described in acute myeloid leukemia (AML). A variety of immunotherapies targeting CD276 including CAR T cells and antibody-drug conjugates (ADCs) are being explored in solid tumors, however their role in leukemia has not been well elucidated. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric and adult AML patients to define the prevalence, frequency of co-occurring genetic alterations and outcomes associated with CD276 expression. Lastly, we evaluated preclinical in vitro efficacy in AML of vobramitamab duocarmazine (vobra duo), an investigational ADC with a humanized B7-H3 mAb conjugated via a cleavable linker to the prodrug seco-DUocarmycin hydroxyBenzamide Azaindole. Ribo-depleted RNA sequencing data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytomolecular aberrations. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients evaluated, CD276 mRNA expression was positive (defined as mRNA transcripts per million (TPM) &amp;gt;2) in 289 patients (20%). Higher prevalence (58%) was seen in adult AML patients (BEAT AML N=209/451, SWOG N=191/199, TCGA-LAML N=85/179), Fig 1A. Flow cytometry validated cell surface expression in pediatric AML, with 22% of patients expressing CD276. Cell surface expression was also observed in AML cell lines. CD276 mRNA expression is absent in normal hematopoietic cells (TPM &amp;lt;1) with absent cell surface expression in normal bone marrow as assessed by flow cytometry, allowing for targeted CD276 therapies to be utilized with no/minimal on-target off-tumor hematopoietic toxicity. Evaluation of co-occurring cytomolecular aberrations demonstrated a significant enrichment of CD276+ pediatric patients with high-risk genetic alterations, most notably KMT2A fusions (N=150/343, 43.7%), KAT6A-CREBBP (N=11/12, 91.6%) and CBFA2T3-GLIS2 (N=22/39, 56.4%). We evaluated the significance of CD276 mRNA expression on clinical outcomes in childhood AML. 5 year event free survival (EFS) for patients with CD276 expression was 35% compared to 44% for patients without CD276 expression (p=0.0022). Overall survival (OS) was 52% for patients with CD276 expression compared to 61% for patients without CD276 expression (p=0.0015). Relapse was seen in 40.5% of patients with CD276 expression compared to 35.8% of patients without CD276 expression and relapse occurred on average earlier in CD276 positive patients (0.89 years vs. 1.06 years, p=0.02). Given the poor clinical outcomes for patients with high CD276 expression, novel targeted therapies are needed for this high risk cohort. With multiple CD276 immunotherapies under clinical evaluation in solid tumors, we tested vobra duo in OCI-AML2, a CD276 positive KMT2A-rearranged AML cell line, which demonstrated robust cytolytic activity with an IC50 less than 1nM (IC50=0.9306 nM) supporting the potential efficacy of CD276 targeted therapies in AML (Fig 1B). In a large cohort of AML patients, we identified CD276 to be overexpressed in 20% of pediatric AML patients and 58% of adult AML patients with a significant enrichment in high risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and CBFA2T3-GLIS2 fusions. Patients with CD276 expression have poor outcomes with significantly worse EFS and OS compared to patients who do not have CD276 expression. With multiple CD276 immunotherapies under clinical evaluation for solid tumors, we hypothesized that targeting CD276 in AML would result in specific cytolytic activity. Vobra duo showed robust in vitro cytolytic activity against CD276 positive AML cells highlighting the need for ongoing preclinical evaluations of CD276 targeted therapies in AML. Given the established safety profile for vobra duo this provides a clear path for rapid translation to clinical use for high risk AML patients.

Engineered tumor-specific T cells using immunostimulatory photothermal nanoparticles

BackgroundAdoptive T cell therapy (ATCT) has been successful in treating hematological malignancies and is currently under investigation for solid-tumor therapy. In contrast to existing chimeric antigen receptor (CAR) T cell and/or antigen-specific T cell approaches, which require known targets, and responsive to the need for targeting a broad repertoire of antigens in solid tumors, we describe the first use of immunostimulatory photothermal nanoparticles to generate tumor-specific T cells. MethodsSpecifically, we subject whole tumor cells to Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT) before culturing with dendritic cells (DCs), and subsequent stimulation of T cells. This strategy differs from previous approaches using tumor cell lysates because we use nanoparticles to mediate thermal and immunogenic cell death in tumor cells, rendering them enhanced antigen sources. ResultsIn proof-of-concept studies using two glioblastoma (GBM) tumor cell lines, we first demonstrated that when PBNP-PTT was administered at a “thermal dose” targeted to induce the immunogenicity of U87 GBM cells, we effectively expanded U87-specific T cells. Further, we found that DCs cultured ex vivo with PBNP-PTT–treated U87 cells enabled 9- to 30-fold expansion of CD4+ and CD8+ T cells. Upon co-culture with target U87 cells, these T cells secreted interferon-ɣ in a tumor-specific and dose-dependent manner (up to 647-fold over controls). Furthermore, T cells manufactured using PBNP-PTT ex vivo expansion elicited specific cytolytic activity against target U87 cells (donor-dependent 32–93% killing at an effector to target cell (E:T) ratio of 20:1) while sparing normal human astrocytes and peripheral blood mononuclear cells from the same donors. In contrast, T cells generated using U87 cell lysates expanded only 6- to 24-fold and killed 2- to 3-fold less U87 target cells at matched E:T ratios compared with T cell products expanded using the PBNP-PTT approach. These results were reproducible even when a different GBM cell line (SNB19) was used, wherein the PBNP-PTT–mediated approach resulted in a 7- to 39-fold expansion of T cells, which elicited 25–66% killing of the SNB19 cells at an E:T ratio of 20:1, depending on the donor. ConclusionsThese findings provide proof-of-concept data supporting the use of PBNP-PTT to stimulate and expand tumor-specific T cells ex vivo for potential use as an adoptive T cell therapy approach for the treatment of patients with solid tumors.

Development of allogenic nonviral RNA-based CAR-NK therapy targeting CDH17 in relapsed/refractory gastrointestinal cancer.

e14539 Background: Gastrointestinal (GI) cancers are commonly fatal after metastasis. The incidence and mortality of GI tumors are high, and the benefits of existing therapies are limited. Cadherin-17 (CDH17) is highly prevalent and specific to GI cancers which has potential for the development of novel treatments for GI cancers. Cell-based immunotherapy using genetically engineered autologous Chimeric Antigen Receptors T (CAR-T) cells has been clinically efficacious for treating hematological malignancies. However, its manufacturing is extremely expensive and lengthy with ~10% failure production rate. Equipping NK cells with CAR may represent a safer and equally effective approach for use in solid tumors as an alternative to autologous CAR-T-cell therapy. Nonviral RNA-based expression of CAR vector in NK cells will provide immediate and multiple accessibility to cancer patients, lower risk of abnormal genetic alteration mediated by viral integration, and potentially limit side effects. This paper demonstrates the development of RNA-CAR-NK targeting CDH17-positive GI cancers. Methods: Ex vivo expanded human peripheral blood NK (Magicell-NK, Medigen, Taiwan) cells were used in this study. To generate anti-CDH17 (C7) CAR-NK cells, mRNA containing a novel CDH17-specific single-chain variable fragment and the 4-1BB costimulatory domain was electroporated to Magicell-NK cell. Flow cytometry analysis was performed to evaluate the cell viability and the expression level of CAR and cytotoxic surface markers of the CAR-NK cells. CDH17 specific cytolytic activity against AGS gastric cancer cell line and the concentration of cytokines released by CAR-NK cells were measured by in vitro cytotoxicity assay and cytokine release assay, respectively. Results: Our results demonstrated efficient electroporation with high-level C7 CAR expression ( &gt; 80%) and optimal CAR-NK cell viability (~60%). Interestingly, 12 h co-culture of C7 CAR-NK cells with AGS gastric cancer cells at 1:1 and 2:1 effector/target (E:T) ratio showed a significantly enhanced antitumor response of C7 CAR-NK cells on AGS-CDH17+ cells but not on AGS-CDH17- cells, indicating CDH17 target-specific cytolytic activity. Moreover, we observed an elevated level of cytokines such as INF-γ and TNF-α released by C7 CAR-NK cells only in co-culture with AGS-CDH17+ cells, confirming the mechanisms behind the CAR-mediated cytotoxicity. Furthermore, these results were supported by an increased level of surface CD107a or LAMP-1 expression in C7 CAR-NK cells during the 12 h of co-culture with AGS-CDH17+ cells at 1:1 and 2:1 E:T ratio. Conclusions: Our findings suggest that a non-viral based CDH17 CAR-NK cell therapy could be a potential allogeneic, off-the-shelf CAR-NK therapy candidate in GI cancer and support the rationale of further investigating it in vivo and clinical trials.

T-cell surface generation of dual bivalent, bispecific T-cell engaging, RNA duplex cross-linked antibodies (dbBiTERs) for re-directed tumor cell lysis.

Genetic engineered Bispecific T-cell engagers (BiTEs) generate potent cytotoxic effects. Alternately, click chemistry engineered, dual specific bivalent Bispecific T-cell engaging antibodies (dbBiTEs) on T-cell surfaces can be generated from parent monoclonal antibodies. We show the formation of dbBiTEs on the surface of T-cells along with the introduction of complementary 2'-OMe RNA 32-mer oligonucleotides allowing duplex formation between antibodies, designated as dbBiTERs. dbBiTERs generated in solution from anti-CEA and anti-CD3 OKT3 antibodies retained specific binding to CEA positive versus CEA negative cancer cells and to CD3 positive T-cells comparable to dbBiTEs. When T-cells were precoated with dbBiTEs or dbBiTERs and mixed with CEA positive versus CEA negative cancer cells, similar dose dependent and specific cytotoxicity were observed in redirected cell lysis assays. On-cell generated dbBiTERs exerted potent cytotoxic responses against CEA positive targets and were localized at the cell surface by immuno-gold EM. In addition, we demonstrate that target and T-cells, each coated separately with complementary 2'OMe-RNA-linked antibodies can be cross-linked by RNA duplex formation in vitro to generate redirected cell lysis. The facile generation of dbBiTERs with specific cytolytic activity from intact antibodies and their generation on-cell offers a new avenue for antigen specific T-cell therapy.

Abstract 1546: Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy

Abstract IL1RAP (Interleukin 1 receptor accessory protein) is differentially expressed in both Ewing sarcoma (EWS) and select hematologic malignancies (CML, AML). Using single domain antibody-like binders generated against IL1RAP to create chimeric antigen receptors (CARs) we demonstrated highly specific cytokine production and cytolytic activity against multiple EWS cell lines in vitro. Preliminary in vivo functionality of IL1RAP CAR-T indicates that additional approaches will be required to increase the efficacy of IL1RAP CAR-T for orthotopic EWS tumor models. To optimize CAR-T cell therapy for EWS, we used model cell lines to define tumor- and stromal-derived factors that must be overcome to obtain effective treatment. The human cell line TC71 (fusion gene positive) was administered both i.v. and i.m. to NSG mice, and subsequently treated with IL1RAP-specifc CAR-T cells. Tumor engraftment was not seen in the i.v. model, however the orthotopic i.m. model in NSG mice was successful, generating tumor and metastatic lesions surrounded by a collagen rich stromal element when CAR-T or control activated T cells were administered. This was not seen in control untreated tumors. Histochemical analysis of i.m. tumors demonstrated that T cells (CD3 positive) were confined to the tumor stromal boundary in proximity to cells of myeloid lineage (CD11b positive). We therefore analyzed cell surface and soluble factors produced by TC71 by flow cytometry, ELISA, and NanoString mRNA panels. TC71 was cultured alone or with interferon-gamma (IFNG), to model the presence of activated T cells and was found to constitutively express transcripts for both canonical and non-canonical chemokines: GPI, MIF, IL-32, TGFβ1; whereas CSF1, CXCL9, CXCL10 and CXCL11 were only induced by IFNG. MIF and TGF-β1 production were confirmed by ELISA. Flow cytometry revealed that numerous CAR-T targets (IL1RAP, CD276, EGFR and FGFR4) were expressed, as were the immune inhibitory molecules: PDL1, PDL2, Galectin-9, and CTLA-4. MHC class I and II and PDL1 were strongly induced by IFNG. We also observed transcripts for collagen related genes in TC71: COL16A1, 3A1, 5A3, LOXL1, P4HB and SERPINH1 both in vitro and in vivo. Our results indicate that tumor surface expression of immune inhibitory signals, PDL-1, PDL-2, CTLA4, as well as the soluble factors MIF, and TGF-b1, must be considered in armoring CAR-T for effective immune destruction of EWS, along with strategies designed to increase the transmigration of T cells through matrix proteins. Moreover, the chemokines produced by tumor alone, as well as those induced by IFNG, indicate a complex network for recruitment of local and bone marrow-derived stromal cells that create a physical as well as cellular barrier to adoptive immunotherapy which needs to be addressed for effective CAR-T therapy of this solid tumor. Citation Format: Rajesh Kumar, Peter Sullivan, Wei Li, Haifeng Zhang, Virginia Hoglund, Lingyang Wang, Yue Zhang, Poul H. Sorensen, Dimiter S. Dimitrov, Rimas J. Orentas. Defining the immune microenvironment in Ewing's sarcoma to potentiate IL1RAP-targeted CAR-T immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1546.

Nanobody Based Tri-Specific Chimeric Antigen Receptor to Treat Acute Myeloid Leukaemia

Background: Chimeric Antigen Receptor (CAR) T-cell therapy represents a paradigm shift in therapy of advanced haematological malignancies in particular Acute Lymphoblastic Leukaemia. We have engineered multi-antigen specific CAR T-cells targeting three acute myeloid leukaemia (AML) associated antigens through incorporation of nanobody binding domains. Target antigens were selected for their broad expression on both on AML blasts as well as a high expression on Leukaemic Stem Cells (LSC). LSCs are a primitive self-renewing population that can contribute to relapse and may be phenotypically distinct from bulk AML blasts within the same patient. Aims: Our multi-antigenic approach thus aims to overcome antigen negative escape, overcome intra-tumour heterogeneity, and clear both LSCs and bulk disease by designing a tandem CAR capable of sensing CD33, CD123 and CLL1 on target cells. Methods: We initially screened libraries of nanobody binders for each antigen in a second generation CAR format. Once lead binders for each antigen were identified, Tandem CAR (TanCAR) structures were designed taking into consideration data on antigen structure from structural prediction servers and published crystal structures, nanobody domain mapping and antigen expression profiles. A TanCAR was then rationally designed comprising of three nanobodies joined by G4S linkers, fused to a hinge spacer, CD8 transmembrane domains, and 41BBz signalling domains. TanCAR T-cell function was compared to that of CAR T-cells targeting single antigens through in vitro co-culture assays with engineered target cells expressing single antigens (n=3). Antigen-specific cytotoxicity and proliferation were measured by flow cytometry assays; IL-2 and IFNy production was assayed by ELISA. Results: The ability of nanobody binding domains to bind individual antigens within the TanCAR format were confirmed using soluble antigen formats with a murine Fc tags via flow cytometry (Fig 1). In co-culture assays against engineered cell lines expressing single antigens (E:T=1:8, 24hrs) the TanCAR demonstrated antigen specific cytolytic activity (median cytotoxicity = 63%, 59%, and 47% against SupT1-CD33, SupT1-CD123, and SupT1-CLL1 respectively) approximately equal to or greater than single antigen targeting nanobody CAR T-cells (Fig 2a). In further co-culture experiments TanCAR T-cells produced high levels of IL-2 and IFNy when co cultured with cell lines expressing CD33 or CD123 only (n=3, median IL-2 = 9237pg/ml and 8190pg/ml, median IFNy 3924pg/ml and 4842pg/ml respectively) compared to SupT1 NT target cells, whereas low level cytokine production was observed with cell lines that express CLL-1 only (Fig 2b). Proliferation data matched this trend with greater levels of antigen specific proliferation observed when TanCAR T-cells were co-cultured with cells expressing CD33 and CD123 (7day co-culture, E:T=1:10, median fold expansion = 22, 8, and 5 fold for TanCAR co-cultured with cell lines expressing CD33, CD123, and CLL1 respectively), compared to antigen negative SupT1 target cells (Fig 2c). Summary/Conclusion: Collectively, our data demonstrate that TanCAR T-cells operate as an OR gate mediating potent reactivity against target cell lines expressing single AML antigens in vitro. We now aim to test for optimal TanCAR formats and potential additive or synergistic responses where TanCAR T cells are co-cultured with AML cell lines in-vivo (Molm14, PDX). Disclosures Ghorashian: Novartis: Honoraria; UCLB: Patents &amp; Royalties; Amgen: Honoraria. Pule:Mana Therapeutics: Other: entitled to share of revenue from patents filed by UCL; UCLB: Patents &amp; Royalties; Autolus: Current Employment, Other: owns stock in and receives royalties, Patents &amp; Royalties.

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.

Abstract 3245: FT819 path to IND: First-of-kind off-the-shelf CAR19 T-cell for B cell malignancies

Abstract Genetic engineering of T cells using a chimeric antigen receptor targeting CD19 antigen (CAR19) is now a well-established treatment of B cell malignancies. While cellular immunotherapies are entering front line treatment, substantial limitations currently hamper the broad application of adoptive T cell therapies in diverse patient population including dysfunctional starting material, lack of product consistency and purity post genetic engineering and inefficient quantity produced for true on-demand availability. FT819 is a first-of-kind off-the-shelf CAR19-T cell product generated from a renewable pluripotent stem cells for large-scale clinical manufacturing. We previously reported the engineering and characterization of the FT819 clonal master cell bank (MCB) derived from a single cell comprising targeted integration of a novel CD19 1XX CAR into the T-cell receptor (TCR) α constant locus to provide optimally regulated CAR expression and elimination of graft versus host (GvH) response. Here we preview the nonclinical study for the original investigational new drug application of FT819. Derived in a manufacturing process analogous to pharmaceutical drug product development, pilot runs from the MCB demonstrated FT819 can be consistently and uniformly manufactured in cGMP compliance, cryopreserved at clinical scale to support off-the-shelf clinical application with greater than 1e5 fold increase in cellular yield from the starting MCB and can be thawed and directly used for facilitated treatment. Repeatedly, FT819 displayed a uniform product profile of ≥95% CAR+, TCR-, CD45+, CD7+ and CD3+ [intracellular] with majority of CD8 T cells expressing CD8β. FT819 global gene expression profile displayed high similarity to primary CAR19-T cells confirming its identity as a T lymphocyte. Functional assessment demonstrated that FT819 possesses potent antigen specific cytolytic activity against leukemia and lymphoma cell lines (p=0.0004). Additional specificity studies demonstrated on-target, off-tumor cytolysis of CD19+ B cells in mixed lymphocyte reaction assay (85% lysis of CD19+ B cells versus &amp;lt; 2% lysis of T cells). Inability of FT819 to produce a GvH response was confirmed in a co-culture assay with anti-TCR crosslinking antibodies. Disseminated leukemia xenograft mouse studies demonstrated the ability of directly thawed and infused FT819 to control tumor growth (p=0.0003 at day 21). In a systemic administered leukemia model FT819 also showed sustained localization in the bone marrow up to 45 days post injection. Ongoing in vivo studies will assess long-term survival and avoidance of GvH disease. Collectively, these studies demonstrate that FT819 is a potent, consistent and uniform CAR19 T cell product and can be effectively and safely used off-the-shelf in the treatment of B cell malignancies with an original Phase 1 clinical trial planned in 2020. Citation Format: Mili Mandal, Raedun Clarke, Sjoukje van der Stegen, Chia-Wei Chang, Yi-Shin Lai, Alec Witty, Mushtaq Husain, Cheng-Jang Wu, Bi-Huei Yang, Chad Dufaud, Gloria Hsia, Helena Shaked, Laurel Stokely, Helen Chu, Mochtar Pribadi, Gilberto Hernandez, Jason ORourke, Alma Gutierrez, Ramzey Abujarour, Tom Lee, Jolanta Stefanski, Juan Zhen, Meilan Wu, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. FT819 path to IND: First-of-kind off-the-shelf CAR19 T-cell for B cell malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3245.

PF438 NKP30‐BASED CHIMERIC ANTIGEN RECEPTOR REDIRECTED HUMAN T LYMPHOCYTES INDUCE EFFECTIVE ANTITUMORAL IMMUNE RESPONSES TO ACUTE MYELOID LEUKEMIA AND MELANOMA

Background:Adoptive cellular therapy (ACT) using chimeric antigen receptor (CAR) redirected human T lymphocytes has achieved remarkable clinical outcome for acute lymphocytic leukemia and also shows great promise for e.g. multiple myeloma. However, suitable antigens for a specific CAR therapy in acute myeloid leukemia (AML) without significant off‐target effects are still warranted as e.g. CD33 and CD123 are also expressed on hematopoietic stem cells (HSC). In contrast, B7H6, a member of the B7 family, is frequently expressed on various cancers including patient‐derived (primary) AML blasts and e.g. melanoma but not found on normal tissues and recognized by the natural killer (NK) cell activating receptor NKp30.Aims:In this study, we therefore evaluated human T cells as well as CRISPR/Cas9 engineered TCR− T cells expressing two different 2nd. generation NKp30 CARs for effective antileukemic immunity to AML in vitro and in vivo using a patient‐derived AML xenograft (PDX) model. Additionally, antitumoral responses of NKp30 CAR T cells to melanoma were examined.Methods:Expression of B7H6 in various AML lines, primary AML samples, solid tumors and primary fibroblasts was examined by RNA‐based qPCR. T cells were polyclonally stimulated and redirected with a CAR containing the extracellular region of the NKp30, a CD8‐ or, alternatively, a IgG1‐spacer, a CD28 costimulatory domain and a CD3ζ signaling domain by retroviral gene transfer. NKp30 CAR+ T cells were antigen‐specifically stimulated with the B7H6 expressing, HLA class I/II negative melanoma cell line MaMel‐86b (courtesy of Dr. C. Wölfel, III. Dept. of Medicine, UMM) and NKp30 expression was determined by flow cytometry. IFN‐γ ELISPOT and cytotoxicity assays were performed to analyze antileukemic responses to different AML samples as well as to Ma‐Mel 86b in vitro. Moreover, NKp30 CAR+ CRISPR/Cas9 engineered TCR− T cells were studied. Biological activity of redirected T cells was tested in an established AML‐PDX NSG mouse model.Results:qPCR assays revealed B7H6 expression at various levels in &gt;95% of primary AML samples tested in addition to e.g. K562 and HL60 myeloid cell lines while no gene bank RNA expression data are reported for healthy tissues. Following transduction and antigen‐specific expansion ≥90% of CD3+ and TCR−CD3− T cells (more CD8+ than CD4+ T cells) expressed both NKp30 CARs. Upon coculture with the B7H6 expressing K562 and HL‐60 cells as well as primary AML samples NKp30‐redirected T cells elicited strong IFN –γ release and exhibited specific cytolytic activity to both leukemia lines and primary AML blasts but not to B7H6 negative U266 cells as shown by 51Cr‐release and colony‐forming cytotoxicity assays. Interestingly, reactivity of NKp30‐CD8‐CD28‐CD3ζ CAR T cells was significantly higher as compared to the other CAR. In addition, NKp30 CAR T cells induced strong responses to MaMel‐86b cells. Moreover, 3rdparty, TCR− NKp30 CAR T cells excluding alloreactivity also elicited potent responses to B7H6. To test for antitumor immunity in vivo, we first examined reactivity of NKp30‐redirected T cells to K562 engrafted into NSG mice and observed reduction of tumor burden. Further studies to evaluate reactivity to primary AML blasts in a PDX model are in progress.Summary/Conclusion:These studies demonstrate that NKp30 CAR T cells and additionally non allogeneic 3rdparty TCR− NKp30 CAR T cells, particularly expressing a CAR with a short spacer, can successfully elicit immunity to AML and also to solid tumors such as melanoma. As the B7H6 is not expressed on CD34+ HSC, this antigen might be a promising target for ACT to AML.

Abstract A028: Memory stem T-cells expressing an optimized CD30-specific chimeric antigen receptor (CAR) efficiently eradicate peripheral T-cell lymphoma in vivo

Abstract Peripheral T-cell lymphomas (PTCL) represent the most aggressive form among non-Hodgkin lymphomas with a very poor prognosis (5-year survival of 30%), demanding innovative novel treatment strategies. Adoptive immunotherapy with chimeric antigen receptor (CAR) T-cells has demonstrated its therapeutic potential in advanced B-cell hematologic malignancies. However, its application to PTCL remains a formidable challenge mainly due to a lack of truly tumor-specific antigens that are not expressed on normal T-cells. Anaplastic large T-cell lymphomas (ALCL) and several other subtypes of PTCL express CD30, which is also expressed by activated normal T-cells but no other healthy tissues. Indeed, brentuximab-vedotin, an anti-CD30 antibody-drug conjugate, has shown some clinical efficacy in PTCL and ALCL patients although duration of responses is short in the majority of cases. Here, we developed a refined CD30-CAR T-cell approach to target CD30+ PTCL as a potential novel therapeutic strategy. We selected a novel targeting domain that is unaffected by soluble CD30 protein (Nagata S et al., Clin Cancer Res 2002) to prevent blockade of the CD30-CAR. Moreover, we optimized the therapy by using memory stem T-cells (TSCM) to promote engraftment and persistence of CD30-CAR T-cells after transfer.TSCM were generated with CD3/CD28 costimulation in presence of IL-7, IL-15 and IL-21 (Alvarez C et al., J Transl Med 2016). On day 2 of culture, cells were transduced with a third-generation lentiviral vector encoding the CD30-41BBz-EGFRt CAR. The cell line Karpas 299 (ALCL) was used as tumor model. Cytotoxicity assay was performed at 24 hours and the tumor cell death was detected by luminiscence. Mice were injected with Karpas 299 tumor cells (2x10^6 cells/mice; iv) and were treated with CD30-CAR TSCM (1x10^7 cells/mice; iv) when the tumor was established (day 10). Mice were followed daily for survival. The presence of CD30-CAR TSCM in lymphoid organs and their expression of immune checkpoint molecules (i.e., TIM-3, LAG-3) were analyzed at the end of the in vivo study by flow cytometry. TSCM were the most prevalent T-cell subset at day 10 of culture (84±3.1% of total cells), and the CD30-CAR expression was 82.8±1.0% in CD4+ TSCM and 85.2±2.0% in CD8+ TSCM. Although CD30 protein was detected in a fraction of activated T-cells in culture, CD30-CAR TSCM could be expanded ex vivo (CD4+ CD30-CAR TSCM: 96.0±3.2 fold expansion; CD8+ CD30-CAR TSCM: 109.0±4.2 fold expansion). CD30-CAR TSCM conferred specific cytolytic activity against Karpas 299 cells in vitro (tumor cell death 5:1 (effector:target) ratio: 70.2±5.1% vs. 0% with untransduced TSCM; p&amp;lt;0.001), which was not blockade by soluble CD30 protein. CD30-CAR TSCM were able to eliminate T-cell lymphoma in vivo (60% survival vs 0% control; p=0.001). In addition, CD30-CAR T-cells (&amp;gt;80% of detected CD4+ and CD8+ T-cells) were still present in lymphoid organs (bone marrow, spleen and lymph nodes) of surviving mice at 50 days after infusion. Tumor-bearing mice also showed CD30-CAR T-cells in their lymphoid organs (80% of total CD4+ and CD8+ T-cells) and in the tumor (67.5% of detected T-cells). Interestingly, a high expression of exhaustion markers TIM-3 (74%±6.8%) and LAG-3 (34%±9.7%) were found on those tumor-infiltrating lymphocytes (TILs). Collectively, our data demonstrate that TSCM cells can be efficiently transduced and ex vivo expanded with a novel CD30-CAR and confer potent antitumor efficacy against CD30+ PTCL in vitro and in vivo, with a potential improvement of the therapy using an immune checkpoint blockade for either TIM-3 or LAG-3. Our findings suggest the potential to improve outcome of patients with CD30+ PTCL through adoptive therapy with CD30-CAR modified T-cells. Citation Format: Laura Escribà-Garcia, Carmen Alvarez-Fernández, Ana Carolina Caballero, Rydzek Julian, Einsele Hermann, Jorge Sierra, Michael Hudecek, Javier Briones. Memory stem T-cells expressing an optimized CD30-specific chimeric antigen receptor (CAR) efficiently eradicate peripheral T-cell lymphoma in vivo [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A028.

Targeting bladder cancer using activated Tcells armed with bispecific antibodies.

In the present study, we aimed to investigate whether EGFR or HER2 may serve as a target for Tcell-mediated immunotherapy against human bladder cancer. Expression of EGFR and HER2 was detected on the surface of bladder cancer cells, including Pumc-91 and T24cells, and their chemotherapeutic drug-resistant counterparts. Activated Tcells (ATCs) were generated from healthy PBMCs that were stimulated by the combination of anti-CD3 monoclonal antibody and anti‑CD28 monoclonal antibody in the presence of interleukin-2 for 14days. The ATCs were then armed with chemically hetero-conjugated anti-CD3xanti-EGFR (EGFRBi-Ab) or anti-CD3xanti-HER2 (HER2Bi-Ab). The specific cytolytic activity of ATCs armed with EGFRBi-Ab or HER2Bi-Ab against human bladder cancer cells was evaluated by lactate dehydrogenase activity assays invitro. In contrast to unarmed ATCs, EGFRBi-Ab-armed ATCs and HER2Bi-Ab-armed ATCs showed increased cytotoxic activity against bladder cancer cells. Moreover, Bi-Ab-armed ATCs expressed higher levels of activating marker CD69 and secreted more IFN-γ, TNF-α and IL-2 than did unarmed ATCs. EGFRBi-Ab- or HER2Bi-Ab-armed ATCs may provide a promising immunotherapy for bladder cancer.

Abstract 4979: Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma

Abstract Background: Multiple myeloma (MM) is a usually fatal malignancy of plasma cells, with no current therapy considered curative. About 15% of patients diagnosed with MM are stratified as high risk with poor treatment outcomes and short (2-3 years) survival from diagnosis. Standard risk patients tend to live longer but undergo chronic and/or high intensity therapy and likely experience a relapsing and remitting disease pattern. Therefore, there is still a considerable unmet need for innovative therapies that improve outcomes in MM. One such approach is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against malignant cells. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. KITE-585 was developed as a CAR T cell immunotherapy product candidate directed against B cell maturation antigen (BCMA). BCMA is nearly ubiquitously expressed on MM cells, plasma cells and subsets of mature B cells, but with limited or absent expression on other tissues. Methods: We generated &amp;gt;50 fully human IgGs directed against BCMA using the BCMA protein as antigen and selection criteria including affinity, cross-reactivity and poly-specificity. Following assessment of the binding of the IgGs to a MM cell line known to express BCMA, &amp;gt;10 IgGs were identified that met the criteria for affinity and selectivity and had a &amp;gt;50-fold binding over background. The 8 IgGs that demonstrated the highest specific binding were then sequence-converted to single-chain variable fragments (scFvs) and incorporated into CARs. Results: In all but one case, human T cells engineered to express these CAR constructs exhibited specific cytolytic activity against MM cell lines (NCI-H929 and MM.1s). These CAR T cells demonstrated killing efficiencies of &amp;gt;95% at effector:target ratios of 1:1 over a 24-hour period. Similarly antigen-specific production of inflammatory cytokines was observed in response to target cell lines in vitro. Assessment of antigen-dependent proliferation over a 5 day period revealed &amp;gt;80% proliferation in the 7 constructs that showed cytolytic activity in vitro. Multiple different anti-BCMA CAR constructs representing distinct epitope binding bins of BCMA were then selected for in vivo evaluation. In two disseminated tumor models of luciferase labeled NCI-H929 or MM.1s cells injected intravenously (i.v.), a single i.v. injection of anti-BCMA CAR T-cells delayed the progression of disease and significantly increased survival when compared to control treatment. Conclusions: The results of these studies highlight the potential of targeting BCMA with adoptive transfer of engineered T cells for the treatment of MM. Given these positive findings, progress towards Phase 1 clinical studies in MM patients with KITE-585 is continuing. Citation Format: Gregor B. Adams, Jun Feng, Atefah Ghogha, Armen Mardiros, Jodi Murakami, Tammy Phung, Ruben Rodriguez, Stuart Sievers, Tassja J. Spindler, Jed Wiltzius, Clare Yarka, Sean C. Yoder, Tony Polverino. Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2017-4979

The synthesis of TNF-alpha conjugates with alendronic acid

Conjugates of recombinant human tumor necrosis factor alpha (TNFα) and alendronic acid linked through the protein sulfhydryl, carboxyl, and amino groups were obtained with crosslinking agents of different types. The conjugation reactions were conducted in solution and on a solid phase. Unlike the conjugation reactions in solution, the method involving immobilization of active components on a hydroxyapatite column was shown to result in the conjugates with a specified stoichiometry and a high degree of homogeneity. The TNFα conjugates retained the specific cytolytic activity and demonstrated the higher affinity to hydroxyapatite, an analogue of the bone mineral matrix, than TNFα.

73. Enhanced Engineering of Chimeric Antigen Receptor (CAR)-Modified T Cells Using Non-Viral Sleeping Beauty Transposition from Minicircle Vectors

Adoptive therapy with T cells that were modified with gamma-retroviral and lentiviral (LV) vectors to express a CD19-specific chimeric antigen receptor (CAR) has shown remarkable efficacy in pilot clinical trials. However, there are persistent concerns with the use of viral vectors with regard to safety, as well as the cost and scale of vector production required for making CAR T-cell therapy available to patients on a global level. In this study, we have refined non-viral Sleeping Beauty (SB) transposition to provide an effective and broadly applicable gene-transfer strategy with superior safety profile. We demonstrate that SB transposition of CAR transgenes from minicircle (MC) DNA vectors enables dramatically improved transposition rates compared to conventional plasmids. MC-derived CD19-CAR transposons display a highly favorable integration profile and confer stable CAR expression that permits potent anti-tumor functions and rapid manufacture of CAR T-cell products.We prepared MC transposon donor vectors encoding a CD19-CAR or eGFP from corresponding parental pT2 plasmids through site specific recombination, and a MC encoding SB100X transposase. Each MC contained only the promotor and gene of interest, and was devoid of antibiotic resistance gene and bacterial origin of replication. We then performed transfections into CD8+ and CD4+ T cells of healthy donors (n=7) and analyzed transposition rate and stability that could be accomplished when CD19-CAR transposon and SB100X transposase were encoded by MCs vs. equimolar amounts of corresponding conventional plasmids. In each donor, we achieved a significantly higher transposition rate with MCs (mean 49.8% on day 14 post transfection) compared to plasmids (mean 12.8%; 4.4-fold difference; p<0,001). Expression of the CD19-CAR was stable over multiple rounds of expansion and several weeks in culture. Importantly, MC transfection was not only more effective, but also substantially less toxic compared to plasmids and on average, a 6-fold higher yield of CAR T cells could be obtained within 14 days of culture without the need for feeder cell expansion. In functional experiments, CD19-CAR T cells modified with our enhanced MC-based SB transposition strategy were equally effective as LV transduced CD19-CAR T cells, and exerted high levels of specific cytolytic activity, cytokine production including IFN-g and IL-2, and productive proliferation after stimulation with CD19+ lymphoma. Moreover, a single administration of SB-modified CD19-CAR T cells leads to complete eradication of systemic lymphoma in a murine xenograft model (NSG/Raji-ffLuc), and was at least equivalently potent as LV transduced CD19-CAR T cells prepared from the same donor. To address safety, we determined the gene copy number (n=5, range 3-8 in CD8+ T cells) and performed a comprehensive genomic insertion site analysis. The data show a close-to-random integration profile of MC-derived CD19-CAR transposons, without preference for highly expressed or cancer related genes. Intriguingly, a significantly higher proportion of SB integrations had occurred in genomic safe harbors compared to LV integrations (7-fold difference; p<0,001), close to the perfect score expected for random integration. In conclusion, we demonstrate the potential to manufacture CAR T cells using virus-free SB-mediated transposition from MC DNA vectors. The superior safety profile, high level stable transposition rate and ease-of-handling MC vectors position our novel approach to become a preferred gene-transfer strategy in advanced cellular and gene-therapy.

Human CD8(+) T cells transduced with an additional receptor bispecific for both Mycobacterium tuberculosis and HIV-1 recognize both epitopes.

Tuberculosis (TB) and human immunodeficiency virus type 1 (HIV‐1) infection are closely intertwined, with one‐quarter of TB/HIV coinfected deaths among people died of TB. Effector CD8+ T cells play a crucial role in the control of Mycobacterium tuberculosis (MTB) and HIV‐1 infection in coinfected patients. Adoptive transfer of a multitude of effector CD8+ T cells is an appealing strategy to impose improved anti‐MTB/HIV‐1 activity onto coinfected individuals. Due to extensive existence of heterologous immunity, that is, T cells cross‐reactive with peptides encoded by related or even very dissimilar pathogens, it is reasonable to find a single T cell receptor (TCR) recognizing both MTB and HIV‐1 antigenic peptides. In this study, a single TCR specific for both MTB Ag85B199‐207 peptide and HIV‐1 Env120‐128 peptide was screened out from peripheral blood mononuclear cells of a HLA‐A*0201+ healthy individual using complementarity determining region 3 spectratype analysis and transferred to primary CD8+ T cells using a recombinant retroviral vector. The bispecificity of the TCR gene‐modified CD8+ T cells was demonstrated by elevated secretion of interferon‐γ, tumour necrosis factor‐α, granzyme B and specific cytolytic activity after antigen presentation of either Ag85B199‐207 or Env120‐128 by autologous dendritic cells. To the best of our knowledge, this study is the first report proposing to produce responses against two dissimilar antigenic peptides of MTB and HIV‐1 simultaneously by transfecting CD8+ T cells with a single TCR. Taken together, T cells transduced with the additional bispecific TCR might be a useful strategy in immunotherapy for MTB/HIV‐1 coinfected individuals.

Anti-CD3 x EGFR bispecific antibody redirects cytokine-induced killer cells to glioblastoma in vitro and in vivo.

The epidermal growth factor receptor (EGFR) is an attractive target for the immunotherapy of EGFR+ tumors. Adjuvant immunotherapy with cytokine-induced killer (CIK) cells may improve progression-free survival rates in patients suffering from cancer. In the present study, we examined the bispecific antibody anti-CD3 x anti-EGFR (EGFRBi-Ab) for its ability to redirect CIK cells to target EGFR-positive glioblastoma. The specific cytolytic activity of CIK cells armed with EGFRBi-Ab against U87MG-luc cells was evaluated by bioluminescent signal generated using luciferase reporter assay which did not alter the surface molecule expression or proliferation ability of U87MG cells. In contrast to unarmed CIK cells, increased cytotoxic activity of EGFRBi-armed CIK cells against the U87MG-luc target was observed at effector/target (E/T) ratios of 5:1, 10:1, and 20:1. Moreover, EGFRBi-armed CIK cells secreted significantly higher levels of IFN-γ, TNF-α, and IL-2 than their unarmed CIK counterpart cells. Furthermore, in glioblastoma xenograft mice, infusion of the EGFRBi-armed CIK cells successfully inhibited the growth of glioblastoma tumors. The invitro and invivo antitumor effects of EGFRBi-armed CIK cells support their clinical use for treatment of glioblastoma in the future.

Natural Killer p46 Controls Hepatitis B Virus Replication and Modulates Liver Inflammation.

Natural killer (NK) cells play an important role in hepatitis B virus (HBV) infection control, and are regulated by a complex network of activating and inhibitory receptors. However, NK cell activity in HBV patients remains poorly understood. The objective of this study was to investigate the phenotypic and functional characteristics of circulating NK cells in patients during different chronic hepatitis B (CHB) infection stages. We investigated NK cell phenotypes, receptor expression and function in 86 CHB patients and 20 healthy controls. NK cells were purified and NK cell subsets were characterized by flow cytometry. Cytotoxic activity (CD107a) and interferon-gamma (IFN-γ) secretion were examined, and Natural Killer p46 (NKP46) blockade and spontaneous NK cell cytolytic activity against K562, HepG2 and HepG2.215 cell lines was studied. Activating NKp46 receptor expression was higher in inactive HBsAg carriers when compared with other groups (p = 0.008). NKp46 expression negatively correlated with HBV DNA (R = -0.253, p = 0.049) and ALT (R = -0.256, p = 0.045) levels. CD107a was higher in immune-activated groups when compared with immune-tolerant groups (p = 0.039). CD107a expression was related to viral load (p = 0.02) and HBeAg status (p = 0.024). In vitro NKp46 blockade reduced NK cell cytolytic activity against HepG2 and HepG2.215 cell lines (p = 0.02; p = 0.039). Furthermore, NK cells from high viral load CHB patients displayed significantly lower specific cytolytic activity against anti-NKp46-loaded K562 targets (p = 0.0321). No significant differences were observed in IFN-γ secretion (p > 0.05). In conclusion, NKp46 expression regulates NK cell cytolytic function. NKp46 may moderate NK cell activity during HBV replication suppression and HBV-associated liver damage and may be critical for NK cell activity during CHB infection.