51Cr Release Assay Research Articles

Restoration of natural killer cell activity by interferon-free direct-acting antiviral combination therapy in chronic hepatitis C patients.

Interferon-free direct-acting antiviral (DAA) therapy is an effective treatment for chronic hepatitis C (CH(C)) patients. Activity of natural killer (NK) cells was reported to be impaired in patients with hepatitis C virus infection. The aim of this study was to examine whether DAA therapy could restore NK activity in patients with CH(C). Direct-acting antiviral therapy was given to 31 CH(C) patients as asunaprevir/daclatasvir (ASV/DCV) (n = 15), ledipasvir/sofosbuvir (n = 7), ombitasvir/paritaprevir/ritonavir (n = 6), or elbasvir/grazoprevir (n = 3). Prior to therapy (0M), at the completion of the therapy (EOT), and at 24weeks after completion (AFTER), NK activity and the frequency of CD56dim NK and CD56bright NK cells in peripheral blood were estimated by Cr release assay and flow cytometry. Statistical analysis was carried out by anova and the Mann-Whitney U-test. In one of the ASV/DCV-treated patients, treatment was stopped 12weeks after initiation of therapy because of viral breakthrough. The anova showed that NK activity significantly improved at EOT (vs. 0M, P < 0.01) and at AFTER (vs. 0M, P < 0.001) in 30 patients with sustained virologic response. It also showed that the frequency of CD56dim NK cells was significantly increased at EOT and at AFTER (vs. 0M, P < 0.05). In addition, the NK activity ratio (AFTER/0M) had no significant difference between patient groups with higher and lower Fibrosis-4 scores. Direct-acting antiviral therapy in CH(C) patients could improve NK activity by increasing the frequency of CD56dim NK cells. Additionally, our results might imply that DAAs therapy could reduce the risk of hepatocarcinogenesis by restoring innate immune responses.

A simple and rapid method for measuring human natural killer cell function in whole blood by anti-NKp30-conjugated nanoparticles

Abstract Natural killer (NK) cells play a central role as innate immune effector cells to virus infected cells and tumor cells. The standard assay to evaluate NK activity is 51Cr release assay using radio-labeled K562 cells, however, new methods using fluorescence-labeled targets or flow cytometry to examine degranulation markers have been developed recently. In addition to cytotoxicity, NK cells enable to produce various cytokines to regulate the adaptive immune responses in infection, inflammation and cancer. Here we generated nanoparticles conjugated with various antibody for NK receptors and examined to stimulate human whole blood with these nanoparticles instead of K562 target cells. We found that various cytokines such as IFN-γ, IL-6, IL-1β and CCL2 were produced by NK cells in whole blood by anti-NKp30-conjugated nanoparticles in dose-dependent manner more than by anti-NKp46 or anti-NKG2D nanoparticles. Production of IFN-γ was detected within 4 hours and reached maximum level at 24 hours after stimulation. IFN-γ production was relatively high at incubation temperature of 37°C, but that was significantly decreased over 39°C. Importantly, we could evaluate the reproducible cytokine production in same donors at another blood collection date because of the stable stimulus of nanoparticles and less variation data by human manipulation for lymphocyte separation. Additionally, cytokine secretion did not correlate with NK cell-mediated lysis of K562 target cells and NK cell numbers in whole blood. These data indicated that this method using NKp30 nanoparticles might be useful for measurement of individual differences of NK function especially correlated in adaptive immune response and inflammation.

Quantification of NK cell activity using whole blood: Methodological aspects of a new test

Natural Killer (NK) cells are essential in the biological fight against cancer and intracellular pathogens, and their level of activity has in many settings been used as a biomarker for a functional immune response. Currently, NK cell activity is measured using either 51Cr-release assays or flow cytometry based assays revealing the cells´ cytotoxic capacity or by stimulating them to produce cytokines. Although very effective, these are cumbersome techniques not suitable for high volume clinical laboratories.Recently, an assay has been introduced to measure NK cell activity in a simple and standardized manner. Following stimulation of NK cells in whole blood with a recombinant protein, it utilizes the concentration of IFNγ released to the plasma as a surrogate marker for NK cell activity. However, whole blood holds several sources of IFNγ which may blur the results and hamper the interpretation of the test. Therefore, the present study aimed at analyzing how specifically the test is measuring the activity of NK cells.Intracellular flow cytometry showed that NK cells, T cells, and Natural Killer T (NKT) cells were producing IFNγ in the assay, however when analyzing the distribution of lymphocytes in the IFNγ-expressing subset, the proportion of NK cells far exceeded the percentage of T-, and NKT cells (p < .0001). Hence, our data indicate that the readout of the test was indicative of the NK cells´ ability to mount a response and thus the results may pave the way for the assay to become applicable in the clinical setting as an estimate of NK cell activity for both diagnostic and prognostic purposes.

High fat diet risk factor for gingival Immunosuppression and cancer

ObjectiveHigh fat diet leading to obesity has been associated with many diseases including cancer. We determined the effect of high fat diet and obesity in the phenotype and function of immune cell infiltrates within different tissues including gingival tissues of mice with wild type and targeted KRAS mutations in the pancreas.Experimental methodsConditional LSL‐KRASG12D and p48‐Cre or PDX‐1‐Cre mice were bred and their offsprings were used for the studies. Mice with both the KrasG12D and Cre allele were designated as mutant mice (KC). Mice with neither the KrasG12D nor the Cre allele were used as wildtype (WT). Mice were fed either a high fat, high calorie diet (HFCD) or a control diet (CD) for 4 to 6 months before they were used for experiments. NK cytotoxicity was determined by 51Cr release assay and cytokine secretion by ELISA.ResultsThe Following profiles were obtained for NK cell cytotoxicity and secretion of IFN‐g from NK cells purified from spleen of KC or WT mice fed either with CD or HFCD (WT(CD)&gt;WT(HFCD)&gt;KC(CD)&gt;KC(HFCD). Immune cells from blood and pancreas exhibited similar pattern of cytotoxicity to those obtained from the spleen but inverse profile of IFN‐g and IL‐6 secretion (WT(CD)&lt;WT(HFCD)&lt;KC(CD)&lt;KC(HFCD) was seen. Mice fed with HFCD when compared to CD had much less NK cells both in WT and KC mice in pancreas, adipose and gingiva.ConclusionMice fed with HFCD exhibited higher inflammatory index when compared to those fed with CD both in WT and KC mice. In addition, numbers and the cytotoxicity of NK cells were severely decreased in pancreas and gingiva of WT and KC mice fed with HFCD. High fat diet exacerbates the progression of neoplastic lesions in KC mice and could be responsible for the oral mucosa pathologies due to decrease function and numbers of key immune effectors.Support or Funding InformationP01 CA163200; PAPD and XO clubThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A water-soluble derivative of propolis augments the cytotoxic activity of natural killer cells

Ethnopharmacological relevancePropolis, a resinous material collected from numerous plants by honeybees, has historically been used as a health-promoting food. Recently, due to its potential anti-tumor effects, use of propolis has been proposed as an adjuvant therapy to chemotherapy; however, the effects of propolis on immune responses remain unclear. Aim of the studyIn this study, we examined the effects of the oral ingestion of propolis on natural killer (NK) cell activity, which is important in immune surveillance against cancer and viral infections. In addition, we assessed the effects of the major components of the water-soluble powder derivative of propolis (WPP). Materials and methodsC57BL/6 (B6) wild-type (WT) and RAG 2-deficient (RAG-/-) mice and BALB/c WT, interferon (IFN)-γ-deficient (IFN-γ–/–), IFN-γ receptor-deficient (IFN-γR-/-) and RAG-/- mice were orally administered WPP or its major components. NK cell populations and cytotoxic activity were then examined by flow cytometry and 51Cr release assay, respectively. ResultsWhile the cytotoxic activity of NK cells was increased following administration of 100 mg/kg/day of WPP for 7 days or 200 or 500 mg/kg/day of WPP for 4 days in WT mice, the proportions of NK cell populations were unaltered. Similar activation of NK cell cytotoxicity was observed when RAG-/-, but not IFN-γ–/– or IFN-γR-/-, mice were orally administered 200 mg/kg/day of WPP for 4 days. Oral ingestion of artepillin C or p-coumaric acid, but not drupanin, augmented NK cell cytotoxicity in a manner similar to WPP and to the mixture of these three components. ConclusionThese results suggest that oral ingestion of WPP enhances NK cell cytotoxic activity, but not proliferation, in a manner dependent on IFN-γ and without the contribution of acquired immune responses. Further, artepillin C or p-coumaric acid, but not drupanin, may be the components responsible for this augmentation of NK cell cytotoxicity. These findings suggest the possible utility of WPP as a therapeutic for prevention of cancer development and against viral infection through NK cell activation.

A Role of NKR-P1A (CD161) and Lectin-like Transcript 1 in Natural Cytotoxicity against Human Articular Chondrocytes.

Normal cartilage cells are susceptible to lysis by NK cells. This phenomenon may play a role in immune cartilage destruction; however, the mechanisms of chondrocyte recognition by NK cells remain poorly understood. Therefore, the aim of this study was to reveal a possible role of NKR-P1A/lectin-like transcript 1 (LLT1) interaction in NK cell-mediated cytotoxicity against normal human articular chondrocytes. Chondrocytes were isolated from articular cartilage obtained during talonavicular joint surgery. PBMC or polyclonal NK cells isolated from normal donors served as effector cells. Cell-mediated cytotoxicity against chondrocytes was evaluated by means of 18-h 51Cr-release assay. Specific mRNA expression was evaluated by classical and quantitative RT-PCR, and proteins were detected by Western blot analysis. We found that lysis of articular chondrocytes by PBMC or polyclonal NK cells was potentiated by stimulation with IL-2. Stimulation of effector cells with IL-2 downregulated mRNA expression of inhibitory NKR-P1A NK cell receptor, and blocking of NKR-P1A with specific mAbs resulted in increased chondrocyte killing. Chondrocytes constitutively expressed LLT1, a ligand of NKR-P1A. LLT1 expression by chondrocytes could be upregulated by IL-1α and TNF. Chondrocyte treatment with IL-1α resulted in their increased resistance to killing by natural cytotoxic cells. This could be reversed by blocking of NKR-P1A. These results show that susceptibility of normal articular chondrocytes to lysis by NK cells is modulated by NKR-P1A/LLT1 interactions. Thus, NKR-P1A/LLT1 interaction might provide some novel target for therapeutic interventions in the course of pathological cartilage injury.

Natural Killer (NK) Cell Assays in Immunotoxicity Testing.

It is well known that natural killer (NK) cells are involved in defense against viruses and some tumors. NK cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes, and granulysin. It is increasingly important to evaluate NK cell function in immunotoxicity testing. NK cell function can be evaluated by determining cytolytic activity against target tumor cells by the 51Cr-release assay and also by determining the number of NK cells in peripheral blood in humans and in the spleen in animals using flow cytometry. Recently, the intracellular levels of perforin, granzymes, and granulysin determined by flow cytometry have also been used in the evaluation of NK cell function. In addition, chemical-induced apoptosis in NK cells also has been applied to evaluate the immunotoxicity of chemicals. This chapter will describe the methods for NK cell assays in immunotoxicity testing.

Monitoring Natural Killer Cell Function in Human Ovarian Cancer Cells of Ascitic Fluid.

Natural killer (NK) cells are the major effectors of the innate immune system when activated resulting in modulation of immune response of the host defense through target cell lysis and secretion of cytokines. Precise functions of NK cells are essential for the treatment outcome of different virus infections and malignant diseases. NK cells impart cytotoxic effect to the target cells lacking MHC class I molecules and thus the final readout of the activity is death of target cells. The NK cell function is evaluated by the 51Cr-release and/or flow cytometry-based assays. In the present protocol, we have determined the activation of NK cells by the liberation of IL-10 and IFNγ, and subsequently its function by enumerating the number of dead tumor cells originally isolated from the ascitic fluid of ovarian cancer patients. The entire assay is based on cells of the healthy donors and patients. Besides determining function, this method is able to demarcate between NK-cell sensitive and insensitive tumor cells. This technique enables researchers to study NK cell functions in healthy donors or in patients to reveal their impact on different malignancies and to further discover new therapeutic strategies.

Efficacy Against Human Prostate Cancer by Prostate-specific Membrane Antigen-specific, Transforming Growth Factor-β Insensitive Genetically Targeted CD8+ T-cells Derived from Patients with Metastatic Castrate-resistant Disease

Current immunotherapy has limited efficacy on metastatic castrate-resistant prostate cancer (mCRPC). We therefore sought to improve the antitumor ability of mCRPC patient-derived CD8+ T-cells by the endowment of specificity to prostate-specific membrane antigen (PSMA) and insensitivity to immunosuppressant molecule transforming growth factor-β (TGF-ß) under the control of herpes simplex virus-1 thymidine kinase. CD8+ T-cells were collected by leukapheresis and cultured in a Food and Drug Administration-approved Cell Processing Work Station. We developed a chimeric antigen receptor retroviral construct using an anti-PSMA chimeric immunoglobulin-T-cell receptor(ζ) gene (PZ1) and dominant negative TGF-ß type II receptor (TßRIIDN), that could induce CD8+ T-cells to be PSMA reactive and insensitive to TGF-ß. Cr51 release assay was performed on PC-3 and PC-3-PSMA. The further antitumor functions of PSMA-specific, TGF-ß insensitive CD8+ T-cells was evaluated using an immunodeficient RAG-1–/– mouse model. We found PSMA-specific, TGF-ß insensitive CD8+ T-cells from mCRPC were expanded with strong expression of PZ1 and thymidine kinase genes, and their growth was not suppressed by TGF-ß. The survival of these cells decreased sharply after treatment with ganciclovir. Treatment of PSMA-specific TGF-ß, insensitive CD8+ T-cells was associated with 61.58% specific lysis on PC-3-PSMA, and significantly suppressed PC3-PSMA tumor compared with the PC3 tumor. A large amount of tumor apoptosis and CD8+ T-cell infiltration were found only in the PC3-PSMA tumor. This study verified that PSMA-specific, TGF-ß insensitive CD8+ T-cells derived from mCRPC patients could be successfully expanded and used to overcome the immunosuppressive effects of the tumor microenvironment to control PSMA-expressing PC in vitro and in vivo. This may provide a promising approach for men with mCRPC who fail androgen deprivation therapy. Patient summaryWe investigated the role of a novel chimeric antigen receptor T-immunotherapy based on autologous metastatic castrate-resistant prostate cancer patient-derived prostate-specific membrane antigen (PSMA)-specific, transforming growth factor-ß insensitive CD8+ T-cells on PSMA-positive prostate cancer. We found that this chimeric antigen receptor T-cells could kill PSMA-positive prostate cancer specifically. The results suggest that this novel immunotherapy treatment is a potential new approach for men with metastatic castrate-resistant prostate cancer.

Broad Impairment of Natural Killer Cells from Operationally Tolerant Kidney Transplanted Patients.

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ+ NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function.

Enhanced CD8+ cytolytic T cell responses in the peripheral circulation of patients with sarcoidosis and non-Löfgren's disease.

The role of CD4+ T cells in the immunopathogenesis of pulmonary sarcoidosis is well-established, while less is known about the phenotype and function of CD8+ cytolytic T cells (CTLs). CD8+ CTLs were explored in peripheral blood and bronchoalveolar lavage (BAL) samples obtained from up to 25 patients with sarcoidosis and 25 healthy controls. The proportion of CTLs was assessed by the expression of cytolytic effector molecules perforin, granzyme B and granulysin in CD8+ T cells, using flow cytometry. Cytolytic function in blood lymphocytes was assessed using a standard 51Cr-release assay. Patients with Löfgren´s syndrome (LS) and an acute disease onset, were compared to non-LS patients with an insidious onset. Higher proportions of peripheral CD8+ CTLs expressing perforin and granzyme B were observed in sarcoidosis compared to healthy controls. Blood CTLs from non-LS patients had significantly higher expression of perforin, granzyme B and granulysin compared to matched BAL, while LS patients maintained lower levels of effector molecules in both compartments. Mitogen-stimulated peripheral lymphocytes from sarcoidosis patients, particularly from the non-LS group, showed a higher target cell lysis compared to controls. These results demonstrated enhanced peripheral CD8+ CTL responses in sarcoidosis, especially in non-LS patients who have an increased risk of chronic disease. Further comprehensive clinical studies are warranted to increase our understanding of CD8+ CTL responses in sarcoidosis.

In vitro cytotoxicity assays for potency evaluation of immune cells prepared for immunotherapy

Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy. Methods Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line. Intra- and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed. Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range. Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1. BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity. Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency. All of the four assays, especially BATDA and CAM assays, showed good intra- and inter-assay reproducibility. Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay. BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells. Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility. Besides, it is a better assay for detecting the cytotoxicity of adherent cells. BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells. Key words: NK cell; Cytotoxicity; BATDA; CAM; CytoTox-Glo; PKH

Characterization of the flow cytometric assay for ex vivo monitoring of cytotoxicity mediated by antigen-specific cytotoxic T lymphocytes

Several non-radioactive methods have widely been utilized to detect antigen-specific cytotoxic T lymphocyte (CTL) responses instead of the classical 51Cr-release assay. These methods include intracellular cytokine staining, major histocompatibility complex-class I tetramers, and the CD107a mobilization assay. However, they do not directly measure target-cell death. In contrast, several attempts have been made to develop the flow cytometric CTL (FC-CTL) assay for evaluation of cytotoxicity. However, further improvement is necessary for it to become standardized. Here, we evaluated the characteristics of the FC-CTL assay based on the uptake of propidium iodide (PI) using target cell lines expressing the green fluorescent protein (GFP). The FC-CTL assay was found to be sensitive enough to detect primary CTL responses. The usage of a pre-established GFP-expressing target cell line facilitated the procedure of the assay, and enabled a clear discrimination between target and effector cells. Time-course analyses demonstrated that PI-stained target cells were detected as early as surface CD107a expression after antigenic stimulation. Thus, the PI/GFP-based FC-CTL assay is sufficiently sensitive to practically detect the early stages of target-cell death, and may have a great potential for becoming a standard tool to measure CTL activity.

Abstract 3699: Activation of NK cells cytotoxicity mediated by alveolar macrophages in the lung against murine melanoma metastases by combined aerosol immunotherapy

Abstract INTRODUCTION: Taking advantage of aerosol for repeated local applications in the lung, we previously demonstrated that combination of Poly(I:C) and CpG-ODN, TLR3 and 9 ligands capable to activate innate effectors, induced a significant antitumor effect against B16 melanoma lung metastases, which correlated with a strong activation of NK cells. We investigated the sequence of events that lead to NK cells activation by aerosolized TLR agonists in the lung. Although NK cells express certain TLRs, controversies exist regarding their direct responsiveness or dependence on macrophages. Alveolar macrophages have several unique features both promoting host defense mechanisms against invading microorganisms, but also establishing an immunosuppressive microenvironment to avoid inflammatory responses to inhaled particles. Our findings, establishing the sequence of events of lung immune cells activation, might represent the basis for the development of combined aerosol therapies that favor NK cell triggering. EXPERIMENTAL PROCEDURE: Alveolar macrophages and NK cells were recovered by bronchoalveolar lavage and spleen of C57BL/6 mice, respectively. Mice i.v. injected with 5×105 B16 cells were treated starting 7 days later every three days with aerosolized Poly(I:C)/CpG-ODN alone or combined to nebulized myeloid derived suppressive cells (MDSC)-depleting RB6-8C5 antibody. Immune cell populations and expression of M1/M2 markers were analysed by flow cytometry and Real Time PCR, respectively, in digested lungs. Cytotoxic activity of NK cells was assessed by 51Cr release assay. RESULTS: In vitro TLR3/TLR9 stimulation induced IFN-γ secretion by naïve NK cells, but an increase in their cytotoxicity was detected only when NK cells were cocultured with alveolar macrophages pretreated with the two agonists. Alveolar macrophages from melanoma lung metastases-bearing mice treated with aerosolized TLR agonists also promoted NK cell cytotoxicity ex vivo. Moreover, naïve alveolar macrophages incubated with activated NK cells from lungs of melanoma metastases-bearing mice aerosolized with TLR9/TLR3 agonists, up-regulated M1- and downregulated M2-related genes, suggesting a bidirectional crosstalk between NK cells and macrophage. Thus, TLR agonists-activated lung macrophages promote NK cell cytotoxicity and, reciprocally, NK cells have the potential to shape the macrophage programming to M1-like phenotype. Our results also indicated that this positive interplay can be further improved by the depletion of myeloid derived suppressive cells that exert a negative effect on macrophage activation. CONCLUSION: This study points to the promise of combinations of immunotherapeutic agents delivered locally by inhalation to promote macrophages-NK cell activation in the lung as a novel strategy to treat lung cancer patients. Citation Format: Michele Sommariva, Valentino Le Noci, Elda Tagliabue, Andrea Balsari, Lucia Sfondrini. Activation of NK cells cytotoxicity mediated by alveolar macrophages in the lung against murine melanoma metastases by combined aerosol immunotherapy [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 3699. doi:10.1158/1538-7445.AM2017-3699

Abstract 47: SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows antitumor activity in uterine and ovarian carcinosarcoma with HER2/neu expression

Abstract Introduction: Carcinosarcoma (CS) is a rare and highly aggressive gynecologic malignancy. The amplification of HER2/neu has been reported to occur in 25%-56% of uterine and ovarian CSs with significant heterogeneity of the expression within epithelial and in-between epithelial and mesenchymal components. The reported high incidence makes HER2/neu an attractive target for new molecularly targeted therapies. We investigated the efficacy of SYD985, (Synthon-Biopharmaceuticals), a novel HER2-targeting antibody-drug conjugate (ADC) composed of a monoclonal antibody (mAb) similar to trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in uterine and ovarian carcinosarcoma. We also compared the anti-tumor activity of SYD985 to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary CS cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments. Methods: Eight primary CS cell lines were evaluated for HER2/neu surface expression by IHC and gene amplification using FISH assays. The cytotoxicity of SYD985 and T-DM1 was evaluated using these eight CS primary cell lines with differential HER2/neu expression (i.e., 0, 1+, and 3+). Antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability and bystander killing experiments were performed using Cr51-release assays, propidium iodide-based and flow cytometry assays, respectively. SYD985 and T-DM1 in vivo activity was also studied in mouse xenograft models. Results: Similar ADCC were induced by SYD985 and T-DM1 when effector cells were present against CS cell lines with different HER2/neu expression. In contrast, SYD985 was significantly more cytotoxic compared to T-DM1 in the absence of the effector cells. SYD985 is 7 to 54 fold more potent than T-DM1 in comparative experiments and unlike T-DM1, it is active against CS demonstrating moderate/low or heterogeneous HER2/neu expression. Specifically, in HER2/neu 0/1+ cell lines the mean IC50’s were 0.060 µg/mL and 3.221 µg/mL for SYD985 vs T-DM1 (p&amp;lt;0.0001) and in HER2/neu 3+ cell lines 0.013 µg/mL and 0.096 µg/mL, (p&amp;lt;0.0001), respectively, (p&amp;lt;0.0001). Unlike T-DM1, SYD985 induced efficient bystander killing of HER2/neu 0/1+ EOC cells admixed with HER2/neu 3+ cells. In vivo studies confirmed that SYD985 is more active than T-DM1 in CS and effective against HER2/neu 3+ xenografts. Conclusions: We demonstrate for the first time that SYD985 is a novel ADC with remarkable activity against CS not only with strong (3+) but also with low (0/1+) HER2/neu expression . Clinical studies with SYD985 in patients harboring chemotherapy-resistant uterine and ovarian CS with low, moderate and high HER2 expression are warranted. Citation Format: Gulden Menderes, Elena Bonazzoli, Stefania Bellone, Jonathan Black, Gary Altwerger, Alice Masserdotti, Francesca Pettinella, Luca Zammataro, Natalia Buza, Pei Hui, Elena Ratner, Babak Litkouhi, Dan-Arin Silasi, Masoud Azodi, Peter Schwartz, Alessandro D. Santin. SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows antitumor activity in uterine and ovarian carcinosarcoma with HER2/neu expression [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 47. doi:10.1158/1538-7445.AM2017-47

Nimotuzumab Induces NK Cell Activation, Cytotoxicity, Dendritic Cell Maturation and Expansion of EGFR-Specific T Cells in Head and Neck Cancer Patients.

Survival benefit and long-term duration of clinical response have been seen using the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody (mAb) nimotuzumab. Blocking EGFR signaling may not be the only mechanism of action underlying its efficacy. As an IgG1 isotype mAb, nimotuzumab’s capacity of killing tumor cells by antibody dependent cellular cytotoxicity (ADCC) and to induce an immune response in cancer patients have not been studied. ADCC-induced by nimotuzumab was determined using a 51Cr release assay. The in vitro effect of nimotuzumab on natural killer (NK) cell activation and dendritic cell (DC) maturation and the in vivo frequency of circulating regulatory T cells (Tregs) and NK cells were assessed by flow cytometry. Cytokine levels in supernatants were determined by ELISA. ELISpot was carried out to quantify EGFR-specific T cells in nimotuzumab-treated head and neck cancer (HNSCC) patients. Nimotuzumab was able to kill EGFR+ tumor cells by NK cell-mediated ADCC. Nimotuzumab-activated NK cells promoted DC maturation and EGFR-specific CD8+ T cell priming. Interestingly, nimotuzumab led to upregulation of some immune checkpoint molecules on NK cells (TIM-3) and DC (PD-L1), to a lower extent than another EGFR mAb, cetuximab. Furthermore, circulating EGFR-specific T cells were identified in nimotuzumab-treated HNSCC patients. Notably, nimotuzumab combined with cisplatin-based chemotherapy and radiation increased the frequency of peripheral CD4+CD39+FOXP3+Tregs which otherwise were decreased to baseline values when nimotuzumab was used as monotherapy. The frequency of circulating NK cells remained constant during treatment. Nimotuzumab-induced, NK cell-mediated DC priming led to induction of anti-EGFR specific T cells in HNSCC patients. The association between EGFR-specific T cells and patient clinical benefit with nimotuzumab treatment should be investigated.

Therapy with transcutaneous administration of imiquimod combined with oral administration of sorafenib suppresses renal cell carcinoma growing in an orthotopic mouse model.

Imiquimod is an imidazoquinoline immune response modifier that is used in antiviral and antiallergic creams. Combination therapy using transcutaneous imiquimod and oral sorafenib was previously demonstrated to reduce the tumor burden of renal cell carcinoma growing cutaneously in a mouse model. In the present study, an orthotopic mouse model was used to investigate whether combined treatment with oral sorafenib and transcutaneous imiquimod inhibited renal cell carcinoma growing in the kidney. Kidneys of female BALB/c mice were orthotopically implanted with RENCA mouse kidney cancer cells, and the mice were transcutaneously treated with cream containing imiquimod and/or with orally administered sorafenib 5 days following cell implantation. Tumor burden and incidence were determined 28 days following the start of therapy. Splenocyte activity was quantified using the 51Cr release assay and the fluorescence-activated cell sorting assay with cluster of differentiation (CD) 4 and CD8 antibodies. Imiquimod, sorafenib and combination therapy were tolerated well. A combination of transcutaneous imiquimod and oral sorafenib inhibited the growth of RENCA tumors in the kidney significantly compared with the control. The 51Cr release assay demonstrated that transcutaneous imiquimod therapy significantly induced the release of 51Cr from RENCA cells compared with the control. The fluorescence-activated cell sorting assay demonstrated that transcutaneous imiquimod therapy induced CD8+ and CD4- splenocytes compared with the control. In summary, the results of the present study demonstrated that combined treatment with transcutaneous imiquimod and oral sorafenib may be a promising strategy for the treatment of patients with renal cell carcinoma.

Antitumor effect of a whole tumor cell vaccine expressing human fibroblast activation protein in murine tumor models.

e14542 Background: Immunosuppressive factors in the tumor microenvironment reduce the therapeutic efficacy of cancer vaccines; therefore, dampening the tumor immunosuppressive environment while activating antitumor immunity should be a useful approach for cancer therapy. Cancer-associated fibroblasts (CAFs) are among the most important cellular components of the tumor microenvironment and play an important role in the development and progression of tumors. Fibroblast activation protein α (FAPα) is a type II transmembrane protein specifically expressed by CAFs in most epithelial cancers. Gene sequence homology between human and murine FAPα is 90%. Heterogeneity of genes in the evolution of the formation of such nuances can be utilized to break immune tolerance, enhance immunogenicity, induce tumour cell autoimmune responses and thus inhibit tumor growth. Methods: Tumor cells were transfected with human FAPα plasmids employing the cationic lipid DOTAP. Its antitumor effects were investigated in three established tumor models. The potential immune mechanisms were investigated through adoptive immunotherapy and 51Cr release assay. The distributions of the immune cells in the tumor microenvironment were detected by immunohistochemical staining and flow cytometry. Results: Our results shown that whole tumor cell vaccine expressing human FAPα significantly inhibit tumor growth and prolong the survival of tumor bearing mice. This antitumor immune response was involved both of cellular and humoral immune responses. FAPα specific neutralizing antibodies were found in the serum of vaccinated mice and CAFs were significantly reduced within the tumors. Furthermore, this vaccine enhanced the infiltration of CD4+ T cells and CD8+ T cells, and suppressed the accumulation of immunosuppressive cells in the tumor microenvironment. Conclusions: These findings suggest that whole tumor cell vaccine expressing human FAPα inhibit tumor growth by producing FAPα specific neutralizing antibodies and CTLs, and targeting tumor cells and CAFs. These observations provide a new strategy for the clinical use of genetically modified tumor cells as cancer vaccines.

Feasibility of cancer vaccine therapy using dendritic cells generated from induced pluripotent stem cells expressing carcinoembryonic antigen.

e14538 Background: We investigated whether genetically modified human dendritic cells (DCs) derived from induced induced pluripotent stem cells (hiPSDCs) expressing carcinoembryonic antigen (CEA) could induce the CEA specific cytotoxic T lymphcytes (CTLs) against cancer cell lines endogenously expressing CEA in vitro model using healthy volunteers. Furthermore, we established in vivo tumor model using the CEA transgenic mice (TGM) as a preclinical experiment. Methods: Human model; we differentiated hiPSDCs from iPSCs of three healthy donors and transduced the CEA cDNA into DCs. Mouse model; we differentiated miPSDCs in CEA TGM and transduced the CEA cDNA into DCs. Results: Human model; the surface marker expression, cytokine secretion, and migratory capacity of hiPSDCs were equivalent to human monocytes derived-DCs (hMoDCs). After 3 cycles of stimulation to autologous peripheral blood mononuclear cells (PBMCs) by genetically modified hiPSDCs, the cytotoxic assay was performed using a 51Cr release assay. The CTLs induced by hiPSDCs-CEA exhibited CEA specific cytotoxic activity against the target cells expressing. Mouse model; in the CEA transgenic mice model, the CTLs generated in mice immunized with miPSDCs-CEA showed CEA specific cytotoxic activity against MC38-CEA. Moreover, in the subcutaneous tumor model, vaccination with miPSDCs-CEA achieved significantly high growth inhibitory effect for the MC38-CEA. No adverse events were observed. Conclusions: This vaccine therapy using genetically modified iPSDCs is promising for clinical application for patients with gastrointestinal cancer patients.

Preclinical data support leveraging CS1 chimeric antigen receptor T-cell therapy for systemic light chain amyloidosis

Background aimsLight chain amyloidosis (AL) is a protein deposition disorder that is a result of a plasma cell dyscrasia, similar to multiple myeloma (MM). Immunotherapy is an attractive approach because of the low burden of disease, but the optimal target for AL is unclear. CS1 and B-cell maturation antigen (BCMA) are two potential targets because they are expressed on normal plasma cells and MM cells. MethodsWe performed a prospective study evaluating bone marrow specimens of 20 patients with plasma cell diseases, 10 with AL and 10 with MM. We evaluated the clonal population of plasma cells for BCMA and CS1 expression. We designed a second-generation CS1 chimeric antigen receptor (CAR) construct, comprising a CS1 antigen-specific scFv, shortened hinge region and CD28 costimulatory domain. Purified central memory T cells were activated and transduced with a lentiviral vector encoding the CS1 CAR. Cytotoxicity was evaluated using 51Cr release assays. Five days after tumor inoculation, NSG mice were injected intravenously with CS1 CAR T cells. ResultsWhereas CS1 is present on the plasma cells of AL patients, we found BCMA expression in AL to be markedly low. CS1 CAR T cells were cytotoxic against CS1 positive tumor cells and induced durable tumor regressions in mice. DiscussionOur work represents a novel application of CS1-directed CAR T cells while revealing that BCMA would not be a suitable target. We expect AL to be particularly susceptible to CAR T-cell therapy because of the low tumor burden in the bone marrow.