Receptor-mediated Cytotoxicity Research Articles

Targeting pentamidine towards CD44-overexpressing cells using hyaluronated lipid-polymer hybrid nanoparticles.

Biodegradable nanocarriers possess enormous potential for use as drug delivery systems that can accomplish controlled and targeted drug release, and a wide range of nanosystems have been reported for the treatment and/or diagnosis of various diseases and disorders. Of the various nanocarriers currently available, liposomes and polymer nanoparticles have been extensively studied and some formulations have already reached the market. However, a combination of properties to create a single hybrid system can give these carriers significant advantages, such as improvement in encapsulation efficacy, higher stability, and active targeting towards specific cells or tissues, over lipid or polymer-based platforms. To this aim, this work presents the formulation of poly(lactic-co-glycolic) acid (PLGA) nanoparticles in the presence of a hyaluronic acid (HA)-phospholipid conjugate (HA-DPPE), which was used to anchor HA onto the nanoparticle surface and therefore create an actively targeted hybrid nanosystem. Furthermore, ionic interactions have been proposed for drug encapsulation, leading us to select the free base form of pentamidine (PTM-B) as the model drug. We herein report the preparation of hybrid nanocarriers that were loaded via ion-pairing between the negatively charged PLGA and HA and the positively charged PTM-B, demonstrating an improved loading capacity compared to PLGA-based nanoparticles. The nanocarriers displayed a size of below 150nm, a negative zeta potential of -35mV, a core-shell internal arrangement and high encapsulation efficiency (90%). Finally, the ability to be taken up and exert preferential and receptor-mediated cytotoxicity on cancer cells that overexpress the HA specific receptor (CD44) has been evaluated. Competition assays supported the hypothesis that PLGA/HA-DPPE nanoparticles deliver their cargo within cells in a CD44-dependent manner.

Abstract 5185: First in human trial of off-the shelf iPS derived anti-GPC3 NK cells for recurrent ovarian clear cell carcinoma with peritoneal dissemination

Abstract Background: The development of chimeric antigen receptor (CAR) T cell therapy has introduced an effective strategy to guide and promote the immune response. Also, gene-engineering NK cells to express an exogenous CAR receptor allows the innate anti-tumor ability of NK cells to be directed against target tumor antigen. However, these autologous applications are limited by toxicities, restricted trafficking and infiltration into tumor, suboptimal persistence, and exhausted status of immune cells that may cause manufacturing failure. One approach to overcome those limitations is the development of “off-the-shelf” iPS-cell sources. The iCAR-ILC-N101 is an allogeneic human leukocyte antigen (HLA)-homozygous induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) CAR-expressing innate lymphoid cells/natural killer cell (ILC/NK), which has both antigen-specific and NK activating receptor-mediated cytotoxicity. The iCAR-ILC-N101 is produced from the established iPSC strain QHJI01s04, and there is theoretically no risk of developing graft-versus host disease because the product dose not have T cell receptor. The product has a relevant living period in the body, thereby has little concern about residual toxicity and reduces systemic side effects by topical treatment. GPC3 is a cancer-specific membranous protein that is expressed in hepatoblastoma, hepatocellular carcinoma and ovarian clear cell carcinoma (OCCC) but is not expressed in normal tissue. OCCC is a relatively rare malignancy and is associated with poor prognosis. Intraperitoneal administration of iCAR-ILC-N101 is expected to show antitumor activity for OCCC patients with peritoneal dissemination that express GPC3 and reduce systemic side effects, thereby ensuring safety and improving therapeutic efficacy. Preclinical study showed that intraperitoneal injection of iCAR-ILC-N101 for GPC3-positive ovarian tumor-bearing immunodeficient mouse model showed suppressed tumor growth. Method: This is a first-in human phase 1 study to evaluate safety, toxicity and efficacy of the iCAR-ILC-N101 in patients with GPC3-positive advanced or recurrent OCCC harboring peritoneal dissemination. Major inclusion criteria include histologically diagnosed GPC3-positive advanced or recurrent OCCC with peritoneal dissemination who are resistant to standard therapy and have matched HLA-A24 or B52. The study includes 3 cohorts (cohort -1, 0.5x106 cells/kg; cohort 1, 1x106 cells/kg; cohort 2, 3x106 cells/kg) and starts with cohort 1. The iCAR-ILC-N101 is administered intraperitoneally once a week for 4 weeks; for the first patient in each cohort, patient is observed for 14 days for safety evaluation after the first administration and then receive iCAR-ILC-N101 on day15 and 22. Enrollment initiated in July 2021 and one patient was enrolled. No dose-limiting toxicity was observed. Clinical trial registry number: jRCT2033200431 Citation Format: Kenichi Harano, Shin Kaneko, Tetsuya Nakatsura, Junichiro Yuda, Nozomu Fuse, Akihiro Sato, Reiko Watanabe, Genichiro Ishii, Toru Mukohara, Hiroshi Tanabe, Yukiko Ishiguro, Hideki Furuya, Masashi Wakabayashi, Miki Fukutani, Manami Shimomura, Tatsuki Ueda, Shoichi Iriguchi, Ayako Kumagai, Kengo Nakagoshi, Aki Sasaki, Toshihiko Doi. First in human trial of off-the shelf iPS derived anti-GPC3 NK cells for recurrent ovarian clear cell carcinoma with peritoneal dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5185.

Dual checkpoint targeting of B7-H3 and PD-1 with enoblituzumab and pembrolizumab in advanced solid tumors: interim results from a multicenter phase I/II trial

BackgroundAvailability of checkpoint inhibitors has created a paradigm shift in the management of patients with solid tumors. Despite this, most patients do not respond to immunotherapy, and there is considerable...

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.

Actively targeted delivery of SN38 by ultrafine iron oxide nanoparticle for treating pancreatic cancer.

Pancreatic cancer remains one of the most lethal cancers largely due to the inefficient delivery of therapeutics. Nanomaterials have been extensively investigated as drug delivery platforms, showing improved drug pharmacodynamics and pharmacokinetics. However, their applications in pancreatic cancer have not yet been successful due to limited tumor delivery caused by dense tumor stroma and distorted tumor vasculatures. Meanwhile, smaller-sized nanomaterials have shown improved tumor delivery and retention in various tumors, including pancreatic tumors, suggesting their potential in enhancing drug delivery. An ultrafine iron oxide nanoparticle (uIONP) was used to encapsulate 7-ethyl-10-hydroxyl camptothecin (SN38), the water-insoluble active metabolite of pancreatic cancer chemotherapy drug irinotecan. Insulin-like growth factor 1 (IGF-1) was conjugated to uIONP as a ligand for targeting pancreatic cancer cells overexpressing IGF-1 receptor (IGF1R). The SN38 loading and release profile were characterized. The pancreatic cancer cell targeting using IGF1-uIONP/SN38 and subsequently induced cell apoptosis were also investigated. IGF1-uIONP/SN38 demonstrated a stable drug loading in physiological pH with the loading efficiency of 68.2 ± 3.5% (SN38/Fe, wt%) and < 7% release for 24h. In tumor-interstitial- and lysosomal-mimicking pH (6.5 and 5.5), 52.2 and 91.3% of encapsulated SN38 were released over 24h. The IGF1-uIONP/SN38 exhibited specific receptor-mediated cell targeting and cytotoxicity Ato MiaPaCa-2 and Panc02 pancreatic cancer cells with IC50 of 11.8 ± 2.3 and 20.8 ± 3.5nM, respectively, but not to HEK293 human embryonic kidney cells. IGF1-uIONP significantly improved the targeted SN38 delivery to pancreatic cancer cells, holding the potential for in vivo theranostic applications.

A Cell-Culture Technique to Encode Glyco-Nanoparticles Selectivity.

Nanoparticles (NPs) embedded with bioactive ligands such as carbohydrates, peptides, and nucleic acid have emerged as a potential tool to target biological processes. Traditional in vitro assays performed under statistic conditions may result in non-specific outcome sometimes, mainly because of the sedimentation and self-assembly nature of NPs. Inverted cell-culture assay allows for flexible and accurate detection of the receptor-mediated uptake and cytotoxicity of NPs. By combining this technique with glyco-gold nanoparticles, cellular internalization and cytotoxicity were investigated. Regioselective glycosylation patterns and shapes of the NPs could tune the receptors' binding affinity, resulting in precise cellular uptake of gold nanoparticles (AuNPs). Two cell lines HepG2 and HeLa were probed with galactosamine-embedded fluorescent AuNPs, revealing significant differences in cytotoxicity and uptake mechanism in upright and invert in vitro cell-culture assay, high-specificity toward uptake, and allowing for a rapid screening and optimization technique.

Design and Implementation of NK Cell-Based Immunotherapy to Overcome the Solid Tumor Microenvironment.

Simple SummaryCell therapy over the last few years has revolutionized the treatment of blood cancers. However, efficacy against cancers of solid organs remains limited. These cancers pose a major challenge to the success of cell therapy by generating a suppressive environment that inhibits immunity. We review current efforts to improve the potency of natural killer (NK) cell therapy in the tumor microenvironment. The successful application of these approaches will lead to more effective treatment options and improve clinical outcomes for patients with solid cancers.Natural killer (NK) cells are innate immune effectors capable of broad cytotoxicity via germline-encoded receptors and can have conferred cytotoxic potential via the addition of chimeric antigen receptors. Combined with their reduced risk of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), NK cells are an attractive therapeutic platform. While significant progress has been made in treating hematological malignancies, challenges remain in using NK cell-based therapy to combat solid tumors due to their immunosuppressive tumor microenvironments (TMEs). The development of novel strategies enabling NK cells to resist the deleterious effects of the TME is critical to their therapeutic success against solid tumors. In this review, we discuss strategies that apply various genetic and non-genetic engineering approaches to enhance receptor-mediated NK cell cytotoxicity, improve NK cell resistance to TME effects, and enhance persistence in the TME. The successful design and application of these strategies will ultimately lead to more efficacious NK cell therapies to treat patients with solid tumors. This review outlines the mechanisms by which TME components suppress the anti-tumor activity of endogenous and adoptively transferred NK cells while also describing various approaches whose implementation in NK cells may lead to a more robust therapeutic platform against solid tumors.

Abstract 4544: Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models

Abstract CD37 is a tetraspanin expressed on mature B cells where it orchestrates plasma membrane organization, receptor signaling, cell migration and adhesion. As CD37 is abundantly expressed on many mature B cell-derived malignancies, it represents an attractive target for new antibody therapies. DuoHexaBody®-CD37 is a novel biparatopic bispecific CD37 antibody with an Fc domain engineered to enhance antibody hexamerization upon binding to CD37 on the plasma membrane. DuoHexaBody-CD37 was shown to induce potent complement-dependent cytotoxicity (CDC) of malignant B-cell lines and primary chronic lymphocytic leukemia and non-Hodgkin lymphoma patient samples (Oostindie et al., Blood 2018 132:4170; van der Horst et al., Blood 2018 132:4179). Here, we demonstrate that the DuoHexaBody-CD37 mechanism of action also encompasses FcγR-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Daudi and Raji cells opsonized with DuoHexaBody-CD37 induced FcγRIIa and FcγRIIIa signaling in luciferase reporter assays. In agreement with efficient FcγR engagement, opsonization with DuoHexaBody-CD37 resulted in dose-dependent ADCC and ADCP by human healthy donor peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs), respectively. In a 4-hour Chromium-51 release assay, DuoHexaBody-CD37 induced ADCC in Daudi cells with PMBCs from 11 out of 12 tested donors (average EC50 of 9.87 [±9.98] ng/mL; maximum kill 20.4 [±9.2]%), and in Raji cells with 1 out of 3 donors. Image- and flow cytometry-based assays with MDMs illustrated that in presence of DuoHexaBody-CD37, Daudi cells were efficiently engulfed by effector cells from 3 different donors (average percentage phagocytic macrophages 29.7 ± 7.6%) resulting in target cell depletion (maximum depletion 75.9 [±18.0]%). Potent anti-tumor activity of DuoHexaBody-CD37 in vivo was reported in a screening approach using B-cell lymphoma patient-derived xenograft (PDX) models with single-mouse treatment groups. Two weekly doses of 5 mg/kg DuoHexaBody-CD37 resulted in strong tumor growth inhibition (tumor stasis or tumor regression) in 3/9 models compared to untreated tumors. Follow-up cohort PDX studies with eight mice per group confirmed potent, dose-dependent anti-tumor activity of DuoHexaBody-CD37 at doses as low as 1 mg/kg. In summary, DuoHexaBody-CD37 induces efficient tumor cell kill through CDC, ADCC and ADCP in vitro and shows potent anti-tumor activity in B-cell lymphoma PDX models in vivo. These data further strengthen the rationale for exploring the safety and efficacy of DuoHexaBody-CD37 in B-cell malignancy patients. Citation Format: Laurens P. Kil, Simone C. Oostindie, Kristin Strumane, Hilma J. van der Horst, Berris van Kessel, Marije B. Overdijk, Andreas Lingnau, Marcel Brandhorst, Jeroen van den Brakel, Margaret A. Lindorfer, Ronald P. Taylor, Martine E. Chamuleau, Tuna Mutis, A. Kate Sasser, Janine Schuurman, Paul W. Parren, Frank J. Beurskens, Esther C. Breij. Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models [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 4544.

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.

The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor

Information about the receptor-interaction region of Cry toxins, insecticidal proteins produced by Bacillus thuringiensis, is needed to elucidate the mode of action of Cry toxins and improve their toxicity through protein engineering. We analyzed the interaction sites on Cry1Aa with ABC transporter C2 (ABCC2), one of the most important Cry1A toxin receptors. A competitive binding assay revealed that the Bombyx mori ABCC2 (BmABCC2) Cry1A binding site was the same as the BtR175 binding site, suggesting that the loop region of Cry1Aa domain II is a binding site. Next, we constructed several domain II loop mutant toxins and tested their binding affinity in an SPR analysis, and also performed a cell swelling assay to evaluate receptor-mediated cytotoxicity. Our results indicate that the loop regions required for BtR175 and BmABCC2 binding and the regions important for cytotoxicity partially overlap. Our results also suggest that receptor binding is necessary but not sufficient for cytotoxicity. This is the first report showing the region of interaction between ABCC2 and Cry1Aa and the cytotoxicity-relevant properties of the Cry1Aa domain II loop region.

New telmisartan-derived PPARγ agonists: Impact of the 3D-binding mode on the pharmacological profile

In previous studies, the 4′-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1′-biphenyl]-2-carboxylic acid was identified as pharmacophoric core for PPARγ activation. In this structure-activity relationship study the C2-alkyl chain was elongated and the 2-COOH group was changed to a carbamide/carbonitrile or shifted to the 3- or 4-position. Furthermore, the benzo[d]imidazole was exchanged by 2,3-dihydrobenzo[d]thiazole or 1H-indole. C2-propyl derivatives showed the profile of partial agonists, while elongation of the C2-chain to that of an n-heptyl group or a 4-COOH shift changed the pharmacological profile to that of a potent full agonist. This finding can be explained by binding to the LBD in different ligand conformations. Two anchoring points (Tyr473 and Arg288) exist in the LBD, which have to be contacted to achieve receptor activation. In a crystal violet chemosensitivity assay using COS-7 cells and LNCaP cells expressing PPARγ only the carbamide derivatives influenced the cell growth, independently on the presence of the PPARγ. Therefore, receptor mediated cytotoxicity can be excluded.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees.

Endogenous expression of a Bt toxin receptor in the Cry1Ac-susceptible insect cell line and its synergistic effect with cadherin on cytotoxicity of activated Cry1Ac

Although many insect cell lines derived from various tissues are available, it is unclear whether endogenous receptors of Bacillus thuringiensis (Bt) crystal toxins are expressed in these cell lines. In the present study, we demonstrated that the ovaries-derived Spodoptera litura Sl-HP cell line was susceptible to activated Cry1Ac although larvae of S. litura are not susceptible to the toxin. Assays of the transcriptome revealed that thirteen ATP-binding cassette transporter genes (ABC) were expressed at different levels in this cell line. Of these, the SlABCC3 shared 52–55% amino acid sequence identity with the known Bt toxin receptor ABCC2. RNAi-mediated knockdown targeting SlABCC3 significantly decreased the susceptibility of Sl-HP cells to activated Cry1Ac. Over-expression of the gene strongly increased the susceptibility of Trichoplusia ni Hi5 cells to the toxin. Not only was SlABCC3 comparable to the heterologously expressed Helicoverpa armigera Hacadherin on the receptor-mediated cytotoxicity of activated Cry1Ac to Hi5 cells, but also SlABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of activated Cry1Ac. These results suggested that Bt toxin receptors-expressing insect cell lines can be used as an alternative model for evaluating cytotoxicity of Bt toxins and studying their mechanisms of action.

Three major uncertainties in the antibody therapy of cancer.

Antibodies against surface molecules of human tumors are now frequently administered in combination with strong chemotherapy, increasing therapeutic efficacy but making the task of elucidating immunological events more difficult. Experiments on genetically manipulated mice indicate that antibody efficacy is greatest when IgG antibody coating tumor cells is engaged by the Fcγ-receptors of effector cells, chiefly the monocyte/macrophage lineage. Evidence suggests lesser roles for NK cells, neutrophils, receptor-mediated cytotoxicity and complement-mediated cytotoxicity. The classical mode of killing employed by macrophages is phagocytosis, but much has to be learned about optimally activating macrophages for this task, and about any other modes of cytotoxicity used. There is renewed interest in antigenic modulation, which implies removal of therapeutic antibody linked with antigen from target-cell surfaces. It is now apparent that this removal of immune complexes can be achieved either by internalization by the target cell, or by transfer of the complexes to another cell by trogocytosis. In trials, anti-idiotype antibodies surprisingly proved therapeutically more effective than anti-CD20, despite anti-idiotype being more effectively removed from target-cell surfaces by antigenic modulation. This anomalous result might reflect the fact that persistence of anti-CD20 immune complexes in large amounts induces serious effector modulation, which paralyzes macrophage attacks on antibody-coated cells. The case for effector modulation is argued by analogy with the therapeutic suppression of autoimmune inflammation by effector modulation, achieved by infusion either of normal IgG in large amounts, or of anti-red cell IgG in relatively small amounts.

Enhanced Receptor-Mediated Endocytosis and Cytotoxicity of a Folic Acid-Desacetylvinblastine Monohydrazide Conjugate in a Pemetrexed-Resistant Cell Line Lacking Folate-Specific Facilitative Carriers but with Increased Folate Receptor Expression

The reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR) are folate-specific transporters. Antifolates currently in the clinic, such as pemetrexed, methotrexate, and pralatrexate, are transported into tumor cells primarily via RFC. Folic acid conjugated to cytotoxics, a new class of antineoplastics, are transported into cells via FR-mediated endocytosis. To better define the role of PCFT in antifolate resistance, a methotrexate-resistant cell line, M160-8, was selected from a HeLa subline in which the RFC gene was deleted and PCFT was highly overexpressed. These cells were cross-resistant to pemetrexed. PCFT function and the PCFT mRNA level in M160-8 cells were barely detectable, and FR-α function and mRNA level were increased as compared with the parent cells. While pemetrexed rapidly associated with FR and was internalized within endosomes in M160-8 cells, consistent with FR-mediated transport, subsequent pemetrexed and (6S)-5-formyltetrahydrofolate export into the cytosol was markedly impaired. In contrast, M160-8 cells were collaterally sensitive to EC0905, a folic acid-desacetylvinblastine monohydrazide conjugate also transported by FR-mediated endocytosis. However, in this case a sulfhydryl bond is cleaved to release the lipophilic cytotoxic moiety into the endosome, which passively diffuses out of the endosome into the cytosol. Hence, resistance to pemetrexed in M160-8 cells was due to entrapment of the drug within the endosome due to the absence of PCFT under conditions in which the FR cycling function was intact.

Dedicator of Cytokinesis 8 Interacts with Talin and Wiskott-Aldrich Syndrome Protein To Regulate NK Cell Cytotoxicity

Recently, patients with mutations in DOCK8 have been reported to have a combined immunodeficiency characterized by cutaneous viral infections and allergies. NK cells represent a first-line defense against viral infections, suggesting that DOCK8 might participate in NK cell function. In this study, we demonstrate that DOCK8-suppressed human NK cells showed defects in natural cytotoxicity as well as specific activating receptor-mediated NK cytotoxicity. Additionally, compared with control NK cells, NK cells depleted of DOCK8 showed defective conjugate formation, along with decreased polarization of LFA-1, F-actin, and cytolytic granules toward the cytotoxic synapse. Using a proteomic approach, we found that DOCK8 exists in a macromolecular complex with the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor activated by CDC42, as well as talin, which is required for integrin-mediated adhesion. Taken together, our results demonstrate an important role for DOCK8 in NK cell effector function and provide important new mechanistic insight into how DOCK8 regulates F-actin and integrin-mediated adhesion in immune cells.

The Parkinson's disease‐associated GPR37 receptor‐mediated cytotoxicity is controlled by its intracellular cysteine‐rich domain

GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that aggregates intracellularly in a juvenile form of Parkinson's disease. However, little is known about the structure or function of this receptor. Here, in order to better understand the functioning of this receptor, we focused on the GPR37 C-terminal tail, in particular on a cystein-enriched region. Thus, we aimed to reveal the role of these residues on receptor plasma membrane expression and function, and also whether the presence of this cysteine-rich domain is linked to the previously described receptor-mediated cytotoxicity. Interestingly, while the deletion of six cysteine residues within this region did not affect receptor internalization it promoted GPR37 plasma membrane expression and signaling. Furthermore, the removal of the C-terminal cysteine-rich domain protected against GPR37-mediated apoptosis and cell death. Overall, we identified a GPR37 domain, namely the C-terminal tail cysteine-rich domain, which played a critical role in receptor cell surface expression, function and GPR37-mediated cytotoxicity. These results might contribute to better comprehend the pathophysiology (i.e. in Parkinson's disease) of this rather unknown member of the GPCR family.

T-Cell Immunotherapy with a Chimeric Receptor Against CD38 Is Effective in Eliminating Myeloma Cells

Abstract 885Myeloma cells themselves are not merely less sensitive to chemotherapy due to their dormancy but prone to becoming drug-resistant. Thus, new therapeutic options are needed for patients with myeloma, especially those over 60 years old. Myeloma cells show the strong expression of CD38. Thus, we investigated whether the retroviral vector-mediated transduction of an anti-CD38-chimeric antigen receptor (CAR) gene contributed to the abrogation of myeloma cells. We had previously designed the retroviral vector carrying anti-CD38-CAR and GFP. T-cells obtained from healthy donors were transduced with retroviral supernatant containing the anti-CD38-CAR vector. The anti-CD38-CAR was expressed with high efficiency. The median percentage of transduced cells was 65.89% (range, 48.98%–89.98%). We confirmed the expression of the anti-CD38-CAR by flow cytometry using a goat-anti-mouse polyclonal antibody conjugated to biotin followed by streptavidin-PerCP, which reacts with the extracellular antibody-derived single-chain variable domain (scFv) of the anti-CD38-CAR. To assess their cytotoxicity, the T-cells transduced with the anti-CD38-CAR were incubated with the KMM1 and RPMI8226 myeloma cell lines for 3 days at a variety of effector (E): target (T) ratios. The transduced cells exerted a highly cytotoxic effect on RPMI8226 cells in a time-dependent manner. The chimeric receptor-mediated cytotoxicity was dose-dependent. The specific cytotoxicity of T-cells with the anti-CD38-CAR to myeloma cell lines at an E: T ratio of 0.5:1. By contrast, there was no detectable cytotoxicity in experiments with the U266-A4 cells, lacking CD38 expression (0.05%). These results demonstrated that CD38-specific T-cells from donors effectively exerted a cytotoxic effect on myeloma cell lines in a dose- and time-dependent fashion. We next evaluated the effect of the anti-CD38-specific T-cells on myeloma cells from the patients. Myeloma cells freshly isolated from the five patients accounted for over 80% of the cells in bone marrow. After myeloma cells were co-cultured with T-cells bearing the anti-CD38-CAR for 3 days, cytotoxic activity was assessed as described above. Intriguingly, mean specific cytotoxicity observed at an E: T ratio of 0.5: 1 for 3 days was >90% in individual patients. Moreover, we obtained the similar results in the autologous settings as well. These results showed that T-cells harboring the anti-CD38-CAR definitively eliminated myeloma cells from the patients as well as myeloma cell lines. Anti-CD38 antibodies or antibody variants have been shown to provide therapeutics for the treatment of CD38-positive hematological malignancies. Introduction of the antibody against CD38 may, however, raise concerns of side effects such as an infusion reaction and pharmacological phenomena associated with the long half-life of the antibody. Although antibodies against CD38 have been administered to patients with myeloma or B-cell lymphoma, there has been no report of adverse effects. Indeed, daratumumab, a novel therapeutic human CD38 antibody and humanized monoclonal CD38 antibody, is currently in clinical trials. In this study, we demonstrated that human T-cells with the anti-CD38-CAR were highly cytotoxic against myeloma cells strongly expressing CD38. Improvements to immunotherapy using autologous T-cells transduced with the anti-CD38-CAR might shed further light on the treatment of myeloma patients with a poor prognosis. Disclosures:No relevant conflicts of interest to declare.

Thermal- and Oxidative Stress Causes Enhanced Release of NKG2D Ligand-Bearing Immunosuppressive Exosomes in Leukemia/Lymphoma T and B Cells

Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

Bombesin Receptor-Mediated Imaging and Cytotoxicity: Review and Current Status

The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered.