Mechanism Of Cell-mediated Cytotoxicity Research Articles

Abstract 2727: Pre-clinical development of a novel anti-GPRC5D inducing potent anti-tumor effect through enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) for multiple myeloma

Abstract Multiple myeloma (MM), characterized by clonal accumulation of malignant plasma cells in the bone marrow, is the second most prevalent hematopoietic malignancy. Despite emerging therapies, MM remains an incurable disease with a 5-year overall survival of 56% for relapsed and/or refractory disease (RRMM) patients. Thus, innovative approaches that would be safe and potent are required. GPRC5D (G Protein-Coupled Receptor Class C Group 5 Member D) is an orphan GPCR protein involved in keratin synthesis with limited expression in healthy tissues. GPRC5D is expressed at very high prevalence among MM patients and its expression on tumor cells is maintained after treatments with standard of care agents such as anti-CD38, or BCMA-targeting therapies. The ability of NK cells to kill tumor cells leaving other cells unharmed and to induce minimal pro-inflammatory cytokine release as compared to T cell-based therapies, makes NK cells ideal immune cells for a safe and efficacious therapeutic approach. Like Sarclisa®, other therapies engaging NK cells and mediating tumor cell killing through antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism have demonstrated their efficacy in clinic. Here we report the design, preclinical characterization, and development of a novel anti-GPRC5D antibody inducing potent cytotoxic activity against MM cells. SAR446523 is an IgG1-based antibody with an engineered Fc domain for enhanced ADCC function and targeting human GPRC5D on tumoral plasma cells. SAR446523 demonstrated high affinity for GPRC5D target and increased affinity for both high and low affinity FCGR3A-158V and 158F variants of CD16a receptor. The ability of SAR446523 to induce ADCC in vitro was assessed in a panel of MM cell lines exhibiting low, medium, and high GPRC5D expression. SAR446523 induced strong potency against all tested cell lines (picomolar range), regardless of the levels of GPRC5D. In addition, SAR446523 induced higher cytotoxic activity compared to a clinical benchmark compound. This anti-tumor activity was associated with very low IL-6, TNFa and IFNg pro-inflammatory cytokine release, much lower compared to a CD3xGPRC5D T-cell engager in in vitro assay using human peripheral blood mononuclear cells (PBMC) in presence of GPRC5D positive cells. In addition, the in vivo anti-tumor activity of SAR446523 was investigated in a NK humanized NOG huIL15 transgenic mouse model bearing disseminated MM cells. SAR446523 led to a robust antitumoral efficacy with a significant improved mouse survival. These data demonstrating potent anti-tumor activity and favorable safety profile of this novel anti-GPRC5D antibody suggest that SAR446523 may be a promising therapy for patients with RRMM and provide consistent support for clinical development. Citation Format: Helene Bonnevaux, Elisabeth Remy, Theo Pizette, Sophie Bouvier, Klervi Desrumeaux, Laurent Bassinet, Celine Nicolazzi, Pauline Rettman, Zandra Klippel, Marco Meloni, Marielle Chiron, Angela Virone-Oddos. Pre-clinical development of a novel anti-GPRC5D inducing potent anti-tumor effect through enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2727.

Escaping Death: How Cancer Cells and Infected Cells Resist Cell-Mediated Cytotoxicity.

Cytotoxic lymphocytes are critical in our immune defence against cancer and infection. Cytotoxic T lymphocytes and Natural Killer cells can directly lyse malignant or infected cells in at least two ways: granule-mediated cytotoxicity, involving perforin and granzyme B, or death receptor-mediated cytotoxicity, involving the death receptor ligands, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL). In either case, a multi-step pathway is triggered to facilitate lysis, relying on active pro-death processes and signalling within the target cell. Because of this reliance on an active response from the target cell, each mechanism of cell-mediated killing can be manipulated by malignant and infected cells to evade cytolytic death. Here, we review the mechanisms of cell-mediated cytotoxicity and examine how cells may evade these cytolytic processes. This includes resistance to perforin through degradation or reduced pore formation, resistance to granzyme B through inhibition or autophagy, and resistance to death receptors through inhibition of downstream signalling or changes in protein expression. We also consider the importance of tumour necrosis factor (TNF)-induced cytotoxicity and resistance mechanisms against this pathway. Altogether, it is clear that target cells are not passive bystanders to cell-mediated cytotoxicity and resistance mechanisms can significantly constrain immune cell-mediated killing. Understanding these processes of immune evasion may lead to novel ideas for medical intervention.

Abstract PR014: A novel immunotherapy for relapsed/refractory pediatric T-cell acute lymphoblastic leukemia

Abstract With the recent addition of nelarabine to first-line therapy, the overall 5-year survival for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients approaches 90%. Patient prognosis for those who relapse is heavily dependent on their specific subtype of ALL: those with B-cell ALL (B-ALL) have several antibody therapies available, including blinatumomab and inotuzumab, which specifically target B-cells for degradation. However, no effective standard therapy is available for patients with relapsed T-cell ALL (T-ALL) and few new therapies are in development; as a result, these patients have a much poorer 5-year survival rate, on the order of 20-30%. Therefore, the development of a targeted immunotherapy for T-cell ALL is critical towards improving patient outcome. The IL-7Ralpha protein is part of the IL-7 receptor complex expressed on the surface of T-cells, and the thymic stromal lymphopoietin (TSLP) receptor complex on the surface of B-cells. The IL-7Ralpha protein is critical for the proliferation and maintenance of T-cells and T-ALL cells, and is overexpressed or constitutively activated in up to 70% of pediatric T-ALL patients. Furthermore, the IL-7Ralpha appears to be overexpressed in chemotherapy-resistant T-ALL cells, highlighting the potential for targeting this protein in relapsed T-ALL patients. Here we describe a novel monoclonal antibody (4A10) raised against the IL-7Ralpha protein which effectively labels T-ALL cells for degradation via antibody-dependent cell-mediated cytotoxicity (ADCC) and independent mechanisms and results in significant control of T-ALL xenografts in animal models, diminished infiltration of T-ALL cells into other organs, and prolonged survival time. The 4A10 antibody is effective against relapsing disease in animal models, and may therefore be effective for relapsed T-ALL patients, or in combination with first-line therapy to further improve initial 5-year survival rates. The 4A10 antibody has undergone extensive optimization and formulation for clinical-grade manufacture and is completing IND-enabling toxicology studies with plans to enter Phase I clinical trials in mid-2021. We are developing a novel clinical trial schema, in collaboration with the Therapeutics Advances in Childhood Leukemia and Lymphoma consortium, to assess tolerability and efficacy via a primarily pediatric Phase I trial, to advance relapsed leukemia patients to curative therapies, including bone marrow transplantation or cell therapies. This abstract is also being presented as PO004. Citation Format: Christopher Foley, Julie Hixon, Wengqing Li, Eric Schafer, Susan Rheingold, Atul Varadhachary, Scott Durum. A novel immunotherapy for relapsed/refractory pediatric T-cell acute lymphoblastic leukemia [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR014.

Real-life rapidity of benralizumab effects in patients with severe allergic eosinophilic asthma: Assessment of blood eosinophils, symptom control, lung function and oral corticosteroid intake after the first drug dose

Benralizumab is a humanized monoclonal antibody which binds to the α subunit of the interleukin-5 (IL-5) receptor and to the FcγRIIIa receptor expressed by natural killer cells, thus suppressing the pro-eosinophil actions of IL-5 and triggering eosinophil apoptosis via the very effective mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). Because of its recent approval and introduction in clinical practice for the add-on biological therapy of severe eosinophilic asthma, real-life investigations are still lacking. In this regard, our present real-life study refers to 13 patients with severe allergic eosinophilic asthma, currently under treatment with benralizumab at the Respiratory Unit of “Magna Græcia” University Hospital located in Catanzaro, Italy.Already 4 weeks after the first subcutaneous injection of benralizumab at the dosage of 30 mg, blood eosinophil count rapidly dropped down from 814.7 ± 292.3 cells/μL to 51.3 ± 97.5 cells/μL (p < 0.0001). This relevant hematologic change was associated with quick and significant increases in asthma control test (ACT) score (from 15.31 ± 2.78 to 21.15 ± 3.58; p < 0.0001), pre-bronchodilator forced expiratory volume in 1 s (FEV1) (from 1441 ± 757.9 mL to 1887 ± 837.3 mL; p < 0.001), and morning peak expiratory flow (PEF) (from 4.21 ± 2.20 to 5.33 ± 1.99 L/sec; p < 0.01). Furthermore, the marked improvement in global health status experienced by our patients allowed them to progressively lower and then completely interrupt, within 4 weeks, their daily intake of oral corticosteroids (OCS), which thereby fell from 15.58 ± 8.30 to 0 mg (p < 0.0001) of prednisone.Therefore, such preliminary results suggest that in patients with severe allergic eosinophilic asthma benralizumab can exert, within a real-life context, a very rapid and effective therapeutic action, already detectable 4 weeks after the first drug administration.

A Novel Fully Human Antibody targeting Extracellular Domain of PSMA Inhibits Tumor Growth in Prostate Cancer.

Prostate cancer is the most commonly diagnosed malignancy in men and the second leading cause of cancer-related death. It is of vital importance to develop new strategies for prostate cancer therapy. PSMA (prostate-specific membrane antigen) is specifically expressed in prostate cancer and the neovasculature of certain cancer types, thus is considered to be an ideal target for cancer therapy. In our previous study, we have obtained a PSMA-specific single-chain variable fragment (scFv), named gy1, from a large yeast display naïve human scFv library. In this study, we reconstructed the PSMA scFv into a fully human antibody (named PSMAb) and evaluated its characterization both in vitro and in vivo We showed that PSMAb can specifically bind with and internalize into PSMA+ cells. The binding affinity of PSMAb is measured to be at nanomolar level, and PSMAb has very good thermostability. In vivo study showed that near IR dye-labeled PSMAb can specifically localize at PSMA+ tumors, and the application of PSMAb in vivo significantly inhibited the growth of PSMA+ tumors, but not PSMA- tumors. At the studied doses, no obvious toxicity was observed when applied in vivo, as shown by the relative normal liver and kidney function and normal structure of important organs, shown by hematoxylin and eosin staining. In addition, PSMAb may inhibit tumor growth through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity mechanisms. Our results indicated that the novel fully human antibody, PSMAb, deserve further study for PSMA-targeted diagnosis and therapy for prostate cancer and other cancer types with vascular PSMA expression.

Mechanisms of natural killer cell-mediated cellular cytotoxicity.

Cellular cytotoxicity, the ability to kill other cells, is an important effector mechanism of the immune system to combat viral infections and cancer. Cytotoxic Tcells and natural killer (NK) cells are the major mediators of this activity. Here, we summarize the cytotoxic mechanisms of NKcells. NKcells can kill virally infected of transformed cells via the directed release of lytic granules or by inducing death receptor-mediated apoptosis via the expression of Fas ligand or TRAIL. The biogenesis of perforin and granzymes, the major components of lytic granules, is a highly regulated process to prevent damage during the synthesis of these cytotoxic molecules. Additionally, NKcells have developed several strategies to protect themselves from the cytotoxic activity of granular content upon degranulation. While granule-mediated apoptosis is a fast process, death receptor-mediated cytotoxicity requires more time. Current data suggest that these 2 cytotoxic mechanisms are regulated during the serial killing activity of NKcells. As many modern approaches of cancer immunotherapy rely on cellular cytotoxicity for their effectiveness, unraveling these pathways will be important to further progress these therapeutic strategies.

Investigation of antitumor activities of trastuzumab delivered by PLGA nanoparticles.

BackgroundWe report the development of an efficient antibody delivery system for the incorporation of trastuzumab (TZ) into poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs). The aim of the work was to overcome the current limitations in the clinical use of therapeutic antibodies, including immunogenicity, poor pharmacokinetics, low tumor penetration and safety issues.Materials and methodsTrastuzumab-loaded PLGA NPs (PLGA-TZ) were synthesized according to a double emulsion method. The same protocol was used to produce control batches of nonspecific IgG-loaded NPs and empty PLGA NPs. After release of TZ from PLGA NPs, the effects on the main biological activities of the antibody were evaluated on SKBR3 (human epidermal growth factor receptor 2 [HER2]-positive breast cancer cell line), including specific binding to HER2, phosphorylation of HER2 (Y1248), degradation of HER2 protein and antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. In addition, an MTT assay was performed for treating SKBR3 cells with PLGA NPs loaded with TZ and doxorubicin to evaluate the cytotoxic activity of the combined treatment.Results and discussionTZ was gradually released in a prolonged way over 30 days. The physical characterization performed with circular dichroism, Fourier transform infrared and fluorescence spectroscopy of TZ after release demonstrated that no structural alterations occurred compared to the native antibody. In vitro experiments using SKBR3 cells showed that TZ released from PLGA NPs maintained the same biological activity of native TZ. PLGA NPs allowed a good co-encapsulation efficiency of TZ and doxorubicin resulting in improved therapy.ConclusionWith the TZ case study, we demonstrate that the distinctive features of therapeutic monoclonal antibodies, including molecular targeting efficiency, capability to inhibit or properly affect the regulatory signaling pathways of cancer cells and stimulation of the ADCC, are fully preserved after loading into and release from PLGA NPs. In addition, PLGA NPs are shown to allow for the simultaneous incorporation of TZ and conventional chemotherapeutics, resulting in a potent antitumor nanodrug well suited for in situ combination and neoadjuvant therapy.

Gene-modified NK-92MI cells expressing a chimeric CD16-BB-ζ or CD64-BB-ζ receptor exhibit enhanced cancer-killing ability in combination with therapeutic antibody.

Natural killer (NK) cells play a pivotal role in monoclonal antibody-mediated immunotherapy through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. NK-92MI is an interleukin-2 (IL-2)-independent cell line, which was derived from NK-92 cells with superior cytotoxicity toward a wide range of tumor cells in vitro and in vivo. Nonetheless, the Fc-receptor (CD16) that usually mediates ADCC is absent in NK-92 and NK-92MI cells. To apply NK-92MI cell-based immunotherapy to cancer treatment, we designed and generated two chimeric receptors in NK-92MI cells that can bind the Fc portion of human immunoglobulins. The construct includes the low-affinity Fc receptor CD16 (158F) or the high-affinity Fc receptor CD64, with the addition of the CD8a extracellular domain, CD28 transmembrane domains, two costimulatory domains (CD28 and 4-1BB), and the signaling domain from CD3ζ. The resulting chimeric receptors, termed CD16-BB-ζ and CD64-BB-ζ, were used to generate modified NK-92MI cells expressing the chimeric receptor, which were named NK-92MIhCD16 and NK-92MIhCD64 cells, respectively. We found that NK-92MIhCD16 and NK-92MIhCD64 cells significantly improved cytotoxicity against CD20-positive non-Hodgkin's lymphoma cells in the presence of rituximab. These results suggest that the chimeric receptor-expressing NK-92MI cells may enhance the clinical responses to currently available anticancer monoclonal antibodies.

Regulation of membrane fixation and energy production/conversion for adaptation and recovery of ZnO nanoparticle impacted Nitrosomonas europaea.

The ZnO nanoparticle (NP) effects on typical ammonia-oxidizing bacteria, Nitrosomonas europaea in a chemostat bioreactor, and the cells' toxicity adaptation and recovery potentials were explored. Hardly any inhibition was observed when the NP concentration was high up to 10mg/L. The cells exposed to 50mg/L ZnO NPs displayed time-dependent impairment and recovery potentials in terms of cell density, membrane integrity, nitrite production rate, and ammonia monooxygenase activity. The 6-h NP stress impaired cells were nearly completely restored during a 12-h recovery incubation, while the longer exposure time would cause irretrievable cell damage. Microarray analysis further indicated the transcriptional adaptation of N. europaea to NP stress. The regulations of genes encoding for membrane permeability or osmoprotectant, membrane integrity preservation, and inorganic ion transport during NP exposure and cell recovery revealed the importance of membrane fixation and the associated metabolisms for cells' self-protection and the following recovery from NP stress. The oxidative phosphorylation, carbon assimilation, and tricarboxylic acid (TCA) cycling pathways involved in the cells' antitoxicity activities and would promote the energy production/conversion efficiency for cell recovery. The heavy metal resistance, histidine metabolism, toxin-antitoxin defense, glycolysis, and sulfate reduction pathways were also suggested to participate in the cell detoxication and recovery processes. All these findings provided valuable insights into the mechanisms of cell-mediated ZnO NP cytotoxicity and their potential impacts on wastewater nitrogen removal system.

Abstract 1496: Identification of target and cytotoxicity of novel monoclonal antibody NEO-201 in ovarian and uterine cancer subtypes

Abstract Objectives: Given the heterogeneity of ovarian cancer, it is imperative to identify subtype-directed treatments. The novel antibody NEO-201 targets a specific tumor-associated antigen (TAA) expressed on some ovarian and uterine malignancies, providing a tumor-directed approach. Here, we aimed to identify the cancer subtypes which express the NEO-201 target and demonstrate its cytotoxic effects in vitro and in mouse models of ovarian cancer. Methods: NEO-201 is a genetically humanized novel monoclonal antibody developed through vaccines with TAAs. This antibody targets malignant tissues that express tumor-specific epitopes in membrane-anchored protein CEACAM-6. We performed immunohistochemistry (IHC) on tissue microarrays from formalin-fixed paraffin-embedded subtyped ovarian and uterine cancers to estimate the incidence of cancers expressing the NEO-201 target. Ovarian and uterine cell line pellet arrays were stained by IHC to identify in vitro models. We examined the cytotoxicity of NEO-201 in two high- and three low-expressing cell lines using Calcein AM cell viability assays alone and with purified natural killer (NK) cells with and without IL-2 stimulation. Studies of NEO-201 in ovarian cancer mouse models are ongoing. Results: IHC of NEO-201 in tissue microarrays demonstrated 51% and 12% reactivity in uterine and ovarian samples, respectively. Similar expression patterns were identified in representative cell lines by IHC and Western blot. NEO-201 killed cell lines expressing its target in the range of 0.5-20μg/mL. Over 3 weeks, NEO-201 treatment of tumor-bearing mice demonstrated control of tumor growth with 3 doses of 250μg NEO-201 and tumor regression with 100μg in combination with IL2-stimulated PBMC. Studies are ongoing to further investigate NEO-201 cytotoxicity in ovarian cancer mouse models as well as identify the mechanism of tumor cell death and the target epitope of NEO-201. Conclusions: NEO-201, a novel monoclonal antibody, specifically targets epithelial malignancies including ovarian and uterine cancer. This potentially therapeutic antibody demonstrates tumor-specific cytotoxicity in cancer cell lines expressing its target in vitro and in xenografts with IL2-stimulated PBMC, suggesting both antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity mechanisms are involved in tumor inhibition and regression. These studies lay the groundwork for future examination of TAA-directed therapy for ovarian or uterine malignancies. Citation Format: Monica K. Neuman, Lidia Hernandez, Xue­-Ping Wang, Olga Saric, Alex Dubeykovskiy, Philip Arlen, Christina M. Annunziata. Identification of target and cytotoxicity of novel monoclonal antibody NEO-201 in ovarian and uterine cancer subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1496.

Antibody immunotherapy targeting the self-tumor antigen nucleolin (VAC5P.1129)

Abstract Nucleolin is a multifunctional protein found in the nucleus of all cells, and it’s a self-tumor antigen. It is overexpressed on the plasma membrane and cytoplasm of the two most common forms of leukemia acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). The cell surface expression of nucleolin on CLL and AML cells, compared with its nuclear profile in normal cells, makes nucleolin an attractive self-antigenic target for antibody immunotherapy. Accordingly, we have utilized our patented platform technology (US Patent 8,715,743) to generate a panel of fully human monoclonal antibodies (HuMAbs) that target nucleolin (NCL) and potently induce AML cell death, while having no effect on normal blood cells (PCT/US2010/057046). Preliminary data indicated that AML cells were killed independently of complement or antibody dependent cellular cytotoxic mechanisms, possibly by interfering with nucleolin’s cellular function. This might have significant therapeutic implications, since the HuMAbs may retain activity in immunocompromised leukemia patients. We hypothesize that the anti-NCL HuMAbs have both immune mediated and immune independent antitumor activity, eliciting nucleolin specific killing through both complement and cell mediated cytotoxic mechanisms, and through interfering with the integral cellular function of nucleolin. Thus, autoreactive antibodies targeting this self-tumor antigen might be important in the treatment of leukemia.

A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells.

The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.

Influence of Btk Inhibitors on Antitumor Activity of Natural Killer Cells

Small molecule Bruton’s tyrosine kinase (Btk) inhibitors are extensively studied in preclinical investigations and clinical trials in the treatment of hematological malignancies derived from B-cells. Ibrutinib, due to its safety and efficiency, has been recently approved for the treatment of patients with Chronic Lymphocytic Leukemia (CLL) who received at least one prior therapy. Moreover, another Btk inhibitor AVL-292 is currently tested in clinical trials in patients with B Cell Non-Hodgkin’s Lymphomas, CLL and Waldenstrom Macroglobulinemia. Despite a huge therapeutic potential of Btk inhibitors we have recently demonstrated that both natural killer (NK) cells cytotoxicity and degranulation are significantly impaired upon ibrutinib treatment (Bojarczuk et al., Leukemia 2014). Since NK cells are effectors of innate immune system capable to kill tumor cells directly and in the mechanism of antibody dependent cell-mediated cytotoxicity (ADCC), which constitutes one of the major mechanism of monoclonal antibodies widely used in hematooncology, in our ongoing studies we are focused to determine in details the influence of various Btk inhibitors on NK cells cytotoxicity, degranulation, cytokine secretion and expression of activatory/inhibitory receptors.All experiments were performed fully in vitro using human primary NK cells isolated from PBMC of healthy donors as well as NK92 and NK92.CD16 cell lines.To determine cytolytic activity of NK cells CFSE/PI flow cytometry assay was used. Cytokine secretion and NK cells degranulation, evaluated as the expression of CD107a on the surface of NK cells, were determined with flow cytometry upon incubation with target cells for 4 h. Phenotype of NK cells pre-incubated with tested compounds was determined with flow cytometry using antibodies conjugated with fluorochromes.The initial results of our studies show that various Btk inhibitors differentially regulate NK cells antitumor activity. Ibrutinib and AVL-292 which covalently bind to cysteines at the position 481 significantly inhibit NK cells cytotoxicity. Interestingly, we have observed that pre-incubation of NK cells with ibrutinib as well as AVL-292 results in down-regulation of CD25 and CD69. This effect was not observed upon treatment of NK cells with CGI-1746, a reversible Btk inhibitor which blocks phosphorylation of BTK in Y551 and Y223. Expression of NK cells activatory receptors such as NKp30, NK44, NKp46, NKG2D, DNAM-1 and CD16 remained unchanged. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NK cell phenotypic and functional heterogeneities and molecular mechanisms of cytotoxicity.

Natural killer (NK) cells were discovered four decades ago, independently, by the Kiessling and Herberman groups. Since then, significant advances have accrued regarding the functional and molecular properties of NK cells as well as their clinical relevance. The important feature of NK cells is their inability to kill normal cells, while they are highly cytotoxic for infected or transformed cells. This discrimination has been largely resolved by the discovery of both activating and inhibitory receptors on NK cells and corresponding ligands on target cells. This review focuses briefly on several issues related to NK cells: (1) the phenotypic properties of NK cells, (2) the heterogeneity of NK cell activities, (3) the determination of three major NK subsets (Free, Binder, and Killer), (4) the maturation and activation of NK subsets, (5) the response of NK cells to various cytokines, (6) the target-induced inactivation and cell death of NK cells, (7) the multiple mechanisms of cell-mediated cytotoxicity, (8) the characterization of NK cytotoxic factors (NKCF), and (9) the role of membrane fluidity in cytotoxicity. These subjects are described chronologically beginning shortly after the discovery of NK cells as a result of Ron Herberman's leadership, and I am gratefully indebted to him.

Immune mechanism and clinical significance of macrophage to medullary hematopoietic injury of immune-related hematocytopenia patients

Background Immune-related hematocytopenia (IRH) is considered to be related with the production of autoantibody, as well as the activation of humoral immunity which is stimulated by B lymphocyte. This study aimed to observe the levels of various cytokines in the blood serum and the in situ active state of macrophage (Mφ) in the medullary hematopoietic microenvironment of IRH patients, and to probe into the immune mechanism and clinical significance of Mφ in hematopoietic cell injury. Methods ELISA is used to detect the IL-4, IL-6, IL-12, IL-17, and IFN-γ levels in the peripheral blood serum of 376 patients in pre- and post-therapy. Cytochemistry and cell immunochemistry methods are used to observe the peroxidase (POX), nonspecific esterase (NSE), hemosiderin granules, and HLA-DR activity of Mφ in the bone marrow of patients. Immunofluorescence is used to observe the expression of hemocyte antihuman globulin IgG antibody, lymphocytes CD4 molecule, Mφ membrane FcγIIreceptor (FcγIIR), mannitose receptor (MR), IFN-γ, ICAM-1, IL-12, and IL-17A and the formation mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC) hematopoietic cell islands (HI) in the medullary hematopoietic microenvironment of patients. Glucocorticoid is used for treatment on the basis of anti-infection therapy, and gamma globulin stoss therapy is used for the appearance of ADCC-type HI or serious Mφ bloodthirsty phenomenon; if necessary, association of Cyclosporine A (CsA) should be used and chalybeate should be supplemented. Results In the patient group, the levels of IL-4, IL-6, IL-12, IL-17, and IFN-γ were increased. After treatment, the cytokine levels gradually became normal. The activated Mφ in the marrow highly expressed NSE and POX, and Mφ swallowed more hemosiderin particles, but the iron in the cytoplasm of immature erythrocytes decreased. The activated Mφ expressed HLA-DR, MR, ICAM-1, IFN-γ, and IL-12. For patients with humoral immunity activation and bacterial infection, Mφ weakly expressed IL-17A but highly expressed FcγIIR, and the phenomenon that ADCC-type HI broke pathological blood corpuscles often occurred; for the cellular immune activation along with virus infection, the white blood count (WBC) significantly reduced, Mφ weakly expressed FcγIIR, secretory highly expressed IL-17A, and the phenomena that Mφ adhered to, captured and swallowed blood cell often occurred. After four weeks of anti-infective and immunosuppressive therapy, nuclear apoptosis of Mφ occurred in the bone marrow of patients, HI and bloodthirsty phenomenon disappeared, and the peripheral blood picture started to improve. Conclusions Mφ is an important antigen presenting cell in the IRH marrow for hematopoiesis destruction and an immune effector cell of hematopoietic injury; infection can promote the activation of Mφ, upregulate the impression of immune molecule and receptors, form ADCC HI, aggravate hematopoietic injury, and accelerate the destruction on hematopoietic cell.

NK Cells Kill Myeloma Cells By Increasing ER Stress and Decreasing Autophagy Levels. NKG2D and NKP30 Are Involved In These Processes

Multiple myeloma (MM) plasma cells are equipped with an extensive endoplasmic reticulum (ER) for antibody production. Misfolding of newly synthesized immunoglobulins leads to ER stress and cell death. Plasma cells evade this by enhancing protein degradation through the proteasome pathway and the autophagy machinery. Because of this, MM cells are sensitive to proteasome inhibitors (PIs). However, autophagy induced by PIs and by reactive oxygen species (ROS) can increase drug resistance and lead to eventual relapse.We have previously demonstrated in vitro and in vivo sensitivity of MM cells to cord blood-derived natural killer (NK) cells, and have observed that this effect may involve NKG2D and NKP30.In this study we used confocal microscopy, chromium cytotoxicity assays and flow cytometry and we aimed to determine if there is a unique mechanism of NK cell-mediated cytotoxicity on MM cells involving ER stress, ROS and its dependence on NK cell receptors NKG2D and NKp30.We first demonstrated a difference in NK-mediated killing of MM cells vs K562 cells. As expected inhibition of GranzymeB (GrB) and Caspase3 pathway yielded a 13% reduction in NK-cytotoxicity against K562 cells; however, a 12% increase in killing was seen against ARP1 cells (p<0.001), indicating a GrB-Caspase3 independent mechanism for NK cell cytotoxicity against ARP1 cells. We observed a decrease in ROS levels after NK treatment for both cell lines (p<0.001), but inhibition of GrB and Caspase 3 abrogated the decrease in ROS only for K562 (p<0.001). By confocal microscopy we observed that NKG2D and NKP30 were transferred to tumor cells and entered the endocytic vesicle pathway. These receptors also appeared to play a more relevant role in the killing of MM cells (33%, 49% and 73% in ARP1 vs 4%, 3% and 19% in K562 cells of killing reduction after blocking NKG2D, NKP30 and both together, p<0.001). Regarding ER stress in ARP1 cells, we saw that after NK treatment there was a significant decrease in endocytic vesicle markers (EEA1 and Rab11, p<0.001) indicating a decrease in the transport of properly folded proteins. There was also a decrease in Rab7 and in Lysosomes (indicated by Lyso-Tracker probes) (p<0.001) and a dramatic increase for DNA-damage-inducible transcript 3 (CHOP) expression (p<0.001) indicating a decreased level of misfolded protein degradation and higher ER stress after exposure to NK cells. Blocking NKG2D abrogated the NK effect on ER stress (indicated by CHOP levels) and ROS levels in ARP1 cells. Blocking of NKP30 abrogated the NK effect on protein degradation activity (Rab7 levels), and ROS levels. To confirm that these receptors mediate killing of ARP1 cells by increasing ER stress, we performed cytotoxicity assays blocking these receptors and adding the PI bortezomib. Bortezomib did not improve NK cell killing at baseline in MM cells. However, bortezomib was able to partially reverse the deleterious effect of blocking NKG2D or NKp30 (33% enhanced killing for NKG2D-blocked and 57% for NKP30-blocked). Finally, we observed that NKG2D and NKP30 were also transferred secondarily to neighboring ARP1 from ARP1 cells which had been primarily synapsed to NK cells (p<0.001). In addition, these secondary ARP1 cells demonstrated decreased ROS levels after this contact. In a cytotoxicity assay, those ARP1 cells which had previously synapsed with NK cells demonstrated 7% of killing against new ARP1 cells.In summary, we describe a new mechanism of killing of MM cells by NK cells mediated by increased ER stress, decreased autophagy and dependent on NKG2D and NKP30. Furthermore, this cytotoxicity can be transmitted between MM cells. These findings could lead to new cellular therapeutic strategies for MM treatment. Disclosures:Shah:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Large Extracellular Loop of Tetraspanin as a Potential Vaccine Candidate for Filariasis

Lymphatic filariasis affects nearly 120 million people worldwide and mass preventive chemotherapy is currently used as a strategy to control this infection. This has substantially reduced the incidence of the infection in several parts of the world. However, a prophylactic vaccine would be more effective in preventing future infections and will supplement the mass chemotherapy efforts. Unfortunately, there is no licensed vaccine available currently to prevent this infection. Molecules expressed on the surface of the parasite are potential candidates for vaccine development as they are exposed to the host immune system. In this study we show that the large extracellular loop of tetraspanin (TSP LEL), a protein expressed on the cuticle of Brugia malayi and Wuchereria bancrofti is a potential vaccine candidate. Our results showed that BmTSP LEL is expressed on the surface of B. malayi infective third stage larvae (L3) and sera from human subjects who are putatively immune to lymphatic filariasis carry high titer of IgG1 and IgG3 antibodies against BmTSP LEL and WbTSP LEL. We also showed that these antibodies in the sera of human subjects can participate in the killing of B. malayi L3 in an antibody dependent cell-mediated cytotoxicity mechanism. Vaccination trials in mice showed that close to 64% protection were achieved against challenge infections with B. malayi L3. Immunized animals showed high titer of anti-WbTSP LEL IgG1, IgG2a and IgG2b antibodies in the sera and IFN-γ secreting cells in the spleen. Onchocerca volvulus another filarial parasite also expresses TSP LEL. Cross-reactivity studies showed that IgG1 antibody in the sera of endemic normal subjects, recognize OvTSP LEL. Similarly, anti-OvTSP LEL antibodies in the sera of subjects who are immune to O. volvulus were also shown to cross-react with rWbTSP LEL and rBmTSP LEL. These findings thus suggested that rTSP LEL can be developed as a potential vaccine candidate against multiple filarial infections.

Eosinophil pathogenicity mechanisms and therapeutics in neuromyelitis optica

Eosinophils are abundant in inflammatory demyelinating lesions in neuromyelitis optica (NMO). We used cell culture, ex vivo spinal cord slices, and in vivo mouse models of NMO to investigate the role of eosinophils in NMO pathogenesis and the therapeutic potential of eosinophil inhibitors. Eosinophils cultured from mouse bone marrow produced antibody-dependent cell-mediated cytotoxicity (ADCC) in cell cultures expressing aquaporin-4 in the presence of NMO autoantibody (NMO-IgG). In the presence of complement, eosinophils greatly increased cell killing by a complement-dependent cell-mediated cytotoxicity (CDCC) mechanism. NMO pathology was produced in NMO-IgG-treated spinal cord slice cultures by inclusion of eosinophils or their granule toxins. The second-generation antihistamines cetirizine and ketotifen, which have eosinophil-stabilizing actions, greatly reduced NMO-IgG/eosinophil-dependent cytotoxicity and NMO pathology. In live mice, demyelinating NMO lesions produced by continuous intracerebral injection of NMO-IgG and complement showed marked eosinophil infiltration. Lesion severity was increased in transgenic hypereosinophilic mice. Lesion severity was reduced in mice made hypoeosinophilic by anti-IL-5 antibody or by gene deletion, and in normal mice receiving cetirizine orally. Our results implicate the involvement of eosinophils in NMO pathogenesis by ADCC and CDCC mechanisms and suggest the therapeutic utility of approved eosinophil-stabilizing drugs.

Comprehensive Investigation of Common Antibody-Dependent Cell-Mediated Cytotoxicity Antibody Epitopes of HIV-1 CRF01_AE gp120

The antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism involves both innate and adaptive immune systems. While a number of epitope mapping studies of neutralizing (Nt) antibodies and cytotoxic T lymphocyte (CTL) against a variety of HIV-1 clades have been reported, there has been a paucity of similar studies aimed at identifying epitopes of ADCC-inducing antibodies. Herein we screened 35 sera from HIV-1 CRF01_AE-infected blood donors for ADCC antibody activity against gp120 utilizing an EGFP-CEM-NK(r) flow cytometric assay. Eighteen sera with high ADCC antibody activity were then comprehensively examined for ADCC antibody epitopes using the HIV-1 subtype CRF01_AE TH023 gp120 peptide set consisting of 126 peptides of 15 amino acids in length, overlapping by 11 amino acids. This peptide set was divided into five pools (E1-E5). Each positive peptide pool was further investigated for fine epitope mapping of ADCC antibody activity using a 5 by 5 peptide matrix format. Interestingly, six and three peptides from peptide pools E1 and E2, respectively, responded to at least 33.33% of the tested sera. These nine common ADCC epitopes were localized to the C1 and V2 region of gp120. Furthermore, 5/9 epitopes were also shown to serve as full or partial Nt antibody targets for HIV-1 subtypes B and CRF01_AE. We submit these data on epitope mapping of ADCC or dual ADCC-Nt antibodies against HIV-1 gp120 that should be considered in the formulation of vaccines against HIV-1.

Favorable Impact of Post-ASCT Consolidative Immunotherapy with RITUXIMAB, rINF-a2b and rIL2 on Overall Survival and Progression-Free Survival in Advanced Stage or Relapsed/Refractory Follicular Lymphoma: Results of a Phase II Study

Abstract 3066 Introduction:Advanced stage III/IV or relapsed/refractory follicular lymphoma (FL) can hardly be cured with conventional chemotherapy and patients, even after autologous stem cell transplantation (ASCT), experience innumerable relapse events occurring at decreasing progression-free intervals. Important progress was made since the introduction of RITUXIMAB as part of frontline chemoimmunotherapy or as maintenance after frontline treatment with or without ASCT. But, whatever the policy applied, no plateau is observed regarding overall survival (OS) and progression-free survival (PFS). Disease relapse, even in patients with documented complete remission (CR), is thought to be induced both by resistant minimal residual tumor cells and the lack of immunocompetent mechanisms to contain or eliminate them. Thus, it has been postulated that post-ASCT consolidative immunotherapy might eradicate minimal residual disease, based on antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism, and have a favorable impact on relapse rate and even OS. This view has led to a considerable interest in immunotherapy as a promising therapeutic line to restore and enhance the impaired anti-tumor immune surveillance in the context of post-ASCT low tumor burden. Purpose:We conducted a phase II study to compare OS and PFS between patients with advanced or relapsed/refractory FL receiving consolidative immunotherapy combining recombinant interleukine-2 (rIL-2), recombinant interferon- α-2b (rINF- α-2b) and RITUXIMAB after ASCT, with the aim of controlling minimal residual disease, and other counterparts with FL receiving no further treatment after ASCT. Patients and Methods:From August, 1995, to October, 2009, 139 patients with FL were autografted in complete remission ≥1 or partial remission. Of these patients, 66 were considered eligible for immunotherapy. On adequate post-ASCT hematologic reconstitution, they received 4 weekly IV injections of RITUXIMAB administered on an outpatient fashion. This was followed by a home-based program of subcutaneous injections of rIL2 (6 million UI × 3 / week in 1st cycle; 9 million × 3 / week in 2d) and rINF- α-2b (1.5 million UI × 3 / week in 1st cycle; 3 million × 3 / week in 2d) administered over two 7-week cycles. These two cycles were separated by a two-week free interval. The other 73 patients did not receive any additional treatment after ASCT and served as controls. Results:The two groups were comparable regarding demographic characteristics, disease profile and treatment course, except that the control group had significantly less transformed FL (p =0.0156) and a higher mean treatment line number (p =0.018). Median OS and PFS were not reached in either the study group or the control group. OS, initially not different between the 2 groups (p =0.21), was found significantly higher in the study group after a cut-off follow-up time point of 86.6 months (p =0.017). A significantly higher PFS was also noted in favor of the study group over the control group (p =0.0131). With a median follow-up of 60 months (10 to 136) in the study group and of 82 months (0.7 to 174) in the control group, survival rate was 90.9% and 71.2%, respectively (p= 0.0034) and the overall relapse rate was 19.7% (13 patients) and 41.1% (30 patients), respectively (p= 0.0064). Adverse events were generally mild, consisting essentially of fever and chills with the rIL-2 and rINF- α-2b subcutaneous injections (rapidly controlled with PARACETAMOL), fatigue, grade 2 abnormal liver function tests in one third of patients and grade 2 or 3 cytopenias (anemia or neutropenia) in 40% of patients. Conclusion:These results confirm that post-ASCT immunotherapy with rIL2, rINF- α-2b and RITUXIMAB has a statistically significant impact on OS and PFS. In view of its tolerability, it appears to be quite feasible and safe and might be seen as a promising line of treatment designed for eradicating post-ASCT minimal residual disease and worth being tested within the frame of a randomized trial for validation as an essential step in the treatment algorithm of FL. Disclosures:No relevant conflicts of interest to declare.