IFN-gamma Induction Research Articles

Development of multivalent vaccine targeting M segment of Crimean Congo Hemorrhagic Fever Virus (CCHFV) using immunoinformatic approaches

Crimean-Congo Hemorrhagic Fever (CCHF) is a tick-borne viral infection with no licensed vaccine or therapeutics available for its treatment. In the present study we have developed the first multi-epitope subunit vaccine effective against all the seven genotypes of CCHF virus (CCHFV). The vaccine contains five B-cell, two MHC-II (HTL), and three MHC-I (CTL) epitopes screened from two structural glycoproteins (Gc and Gn in M segment) of CCHFV with an N-terminus human β-defensin as an adjuvant, as well as an N-terminus EAAAK sequence. The epitopes were rigorously investigated for their antigenicity, allergenicity, IFN gamma induction, anti-inflammatory responses, stability, and toxicity. The three-dimensional structure of the vaccine was predicted and docked with TLR-3, TLR-8, and TLR-9 receptors to find the strength of the binding complexes via molecular dynamics simulation. After codon adaptation, the subunit vaccine construct was developed in a pDual-GC plasmid and has population coverage of 98.47% of the world's population (HLA-I & II combined). The immune simulation studies were carried out on the C-ImmSim in-silico interface showing a marked increase in the production of cellular and humoral response (B-cell and T-cell) as well as TGFβ, IL-2, IL-10, and IL-12 indicating that the proposed vaccine would be able to sufficiently provoke both humoral and cell-mediated immune responses. Thus, making it a new and promising vaccine candidate against CCHFV.

Using Immunoinformatics to Design a mRNA Vaccine against the Spike Glycoprotein of SARS-CoV-2

The rapid outbreak of the new coronavirus SARS-COV-2 has created a major public health challenge. Immunoinformatics tools had a clear effect in tracking the genetic sequence of the virus and monitoring mutations and design vaccines that are effective enough to produce antibodies. In our study, we resorted to the emerging discipline of immunoinformatics in order to design a multi-epitope mRNA vaccine against the spike glycoprotein of SARS-CoV-2. We screened the B cell and T cell epitopes of the Spike glycoprotein. we used ABC pred server to predict B cell epitope in the spike glycoprotein sequence and we used NetMHC-4.1 server to predict the T-cell epitope. Then we selected the B cell and T cell epitopes that fulfilled the antigenicity, non-toxicity, non-allergenicity, induction of both IL4 and IFN gamma. Finally, we designed multi-epitope mRNA Vaccine construct by linking 6 B lymphocytes epitopes (BL) with 6 cytotoxic T lymphocytes epitopes (CTL) together with helper T lymphocyte (HTL) epitope up-streamed by 5’ cap and down-streamed by poly A tail. The vaccine was found to be antigenic, non-toxic, non-allergenic, capable of generating a robust immune response. Based on these parameters, this design can be considered a promising choice for a vaccine against SARS-CoV-2.

Abstract LB048: An adjuvant personalized neoantigen peptide vaccine for the treatment of malignancies (PGV-001)

Abstract Background: The majority of novel cancer immunotherapies rely on adequate priming of T cells to tumor-specific neoantigens, which is believed to be lacking in patients who do not respond to therapy. We developed a personalized genomic vaccine (PGV-001) in which patient-specific synthetic neoantigen peptides (25 mer),are formulated and administered to patients with multiple cancer types in the adjuvant setting (NCT02721043). Methods: This trial enrolled patients whom had undergone curative-intent surgery (solid tumor patients) or autologous stem cell transplant (multiple myeloma patients), and for whom there was >30% chance of recurrence. Sequencing of tumor and germline DNA and RNA was performed and the OpenVax custom computation pipeline was used to identify candidate neoantigens; this platform ranks transcribed mutations using predicted MHC-I binding affinity and neoantigen abundance. A maximum of 10 peptides were synthesized per patient. Peptides were administered over the course of 27 weeks with poly-ICLC and a tetanus helper peptide. Primary objectives were to determine the safety and tolerability of vaccination, feasibility of vaccine production and administration, and immunogenicity. Results: Within 15 patients enrolled, the OpenVax pipeline identified an average of 67.1 neoantigens/patient (range 8-193), only two patients did not have adequate number of neoantigens identified to synthesize 10 peptides. 13 of the 15 patients received PGV-001, including 10 patients with solid tumor diagnoses and 3 patients with multiple myeloma, 11 of whom received all 10 doses, while 1 experienced progression of disease while on treatment. The vaccine was well tolerated, with grade 1 injection site reactions in 31% of patients, and grade 1 fever in one patient; there were no other significant adverse events. While one patient was lost to follow-up, of the remaining 12 patients the median progression-free survival from the time of their surgery or transplant of 618 days. With a mean follow-up of 925 days, 4 patients remain without evidence of disease, 4 patients are receiving subsequent lines of therapy, and 4 patients have died, though notably only two with documented recurrence of their malignancy. Initial analysis of the patient samples analyzed confirms immunogenicity. T cell responses were measured using ex vivo ELISpot and intracellular cytokine staining following expansion with neoantigen peptide libraries, both demonstrating induction of IFN-gamma, TNF-alpha and IL-2. Notably, robust T cell reactivity was only seen at the completion of all 10 vaccines, supporting the need for a prolonged schedule. Conclusions: PGV-001 was successfully synthesized for 15 patients and administered successfully to 13 patients without significant adverse events. Immune monitoring of immunogenicity is ongoing, with initial analysis demonstrating induction of neoantigen-specific CD4 and CD8 T cell expansion. Citation Format: Thomas Urban Marron, Mansi Saxena, Nina Bhardwaj, Marcia Meseck, Alex Rubinsteyn, John Finnigan, Julia Kodysh, Ana Blazquez, Tim O'Donnel, Mathew Galsky, Deborah Doroshow, Brett Miles, Krysztof Misiukiewicz, Hanna Irie, Amy Tiersten, Samir Parekh, Marshall Posner, Andrea Wolf, John Mandeli, Rachel Brody, Sacha Gnjatic, Eric Schadt, Philip Friedlander, Jeffrey Hammerbacher. An adjuvant personalized neoantigen peptide vaccine for the treatment of malignancies (PGV-001) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB048.

Neutrophil Activation Through High-Affinity Fey Receptor Using a Monomeric Antibody With Unique Properties

The high-affinity, type I Ig Fc receptor (Fc gamma RI) for human IgG1, human IgG3, murine IgG2a, and murine IgG3 is highly expressed on monocytes, neutrophils (PMN) in certain disease states, and phagocytes treated with interferon-gamma (IFN-gamma). We studied the activation of the human PMN oxidative burst and stimulated fluid pinocytosis induced by three monoclonal antibodies (MoAbs) directed against Fc gamma RI (CD64) to study the role of this receptor in Fc-mediated cellular activation. All three MoAbs were capable of triggering PMN activation from IFN-gamma-treated PMN when cross-linked with goat antimurine Ig reagents. However, MoAb 197 alone demonstrated a concentration-dependent activation of the oxidative burst without the use of a second cross-linking antibody. The oxidative burst and stimulated fluid pinocytosis responses induced by monomeric MoAb 197 could be elicited only after the IFN-gamma induction of approximately 8,000 Fc gamma RI receptor equivalents and did not occur in freshly isolated or control-cultured PMN. Competitive blocking of Fc binding of MoAb 197 by human IgG or purified Fc fragments inhibited cellular activation. We believe the ability of MoAb 197 to activate these oxidative burst and fluid pinocytic responses was because of its murine IgG2a subclass, which allowed it to function as a trivalent anti-Fc gamma RI antibody binding through the combination of its two FAB regions and the Fc domain. This study demonstrates that the cross-linking of CD64 can activate PMN oxidative and endocytic responses and supports a role for Fc gamma RI in the human neutrophil inflammatory response.

Immunoinformatic construction of an adenovirus-based modular vaccine platform and its application in the design of a SARS-CoV-2 vaccine

The current SARS-CoV-2 pandemic has imposed new challenges and demands for health systems, especially in the development of new vaccine strategies. Vaccines for many pathogens were developed based on the display of foreign epitopes in the variable regions of the human adenovirus (HAdV) major capsid proteins (hexon, penton and fiber). The humoral immune response against the HAdV major capsid proteins was demonstrated to play a role in the development of an immune response against the epitopes in display. Through the immunoinformatic profiling of the major capsid proteins of HAdVs from different species, we developed a modular concept that can be used in the development of vaccines based on HAdV vectors. Our data suggests that different immunomodulatory potentials can be observed in the conserved regions, present in the hexon and penton proteins, from different species. Using this modular approach, we developed a HAdV-5 based vaccine strategy for SARS-CoV-2, constructed through the display of SARS-CoV-2 epitopes indicated by our prediction analysis as immunologically relevant. The sequences of the HAdV vector major capsid proteins were also edited to enhance the IFN-gamma induction and antigen presenting cells activation. This is the first study proposing a modular HAdV platform developed to aid the design of new vaccines by inducing an immune response more suited for the epitopes in display.

ET-11 ANALYSIS OF ANTI-GLIOMA EFFECT BY PROINFLAMMATORY CYTOKINES

OBJECTAntiglioma activity of proinflammatory cytokines, (TNF-alpha, IL-2, IL-12 related cytokines, IL-18, IL-32) are analyzed. Most effective combinations of cytokines are investigated.MATERIAL & METHODAntitumor activity against U373MG, U87MG were measured by co-culture with PBMC and by nude mouse subcutaneous transplantation model. Cytokine receptors on PBMC and glioma cell lines were examined by IHC and mRNA expression. Anti-tumor activity was measured by local injection and systemic administration of proinflammatory cytokines. Cell cycle alteration and expression of apoptosis-related genes after cytokine administration was analyzed. Serum concentraion of cytokines is measured by ELISA.RESULTCytokine receptors were not expressed on glioma cells but were present on intratmoral mononuclear cells. Anti-tumor activity against transplanted tumor is strongly observed by focal administration. Expression of apoptosis-related genes were augmented. IFN-gamma was strongly induced by TNF-alpha, IL-2 and IL-12 administration. IFN-gamma, IL-17, TNF-alpha were also induced. IL-27 and IL-32 per se did not induce IFN-gamma. Simultaneous IL-27 and IL-12 induced strong IFN-gamma induction. Anti-glioma activity of IL-12 and IL-23 were higher than the same dose of exogenous IFN-gamma. IFN-gamma, IL-2 plus IL-12 in U373MG, and IFN-gamma, IL-2 plus IL-18 in U87MG seemed to be the best combination.CONCLUSIONSStrong anti-glioma activity was induced by proinflammatory cytokines at least partially through IFN-gamma. There may be another factors. IL-2 and IL-23 showed anti-tumor activity through IFN-gamma, IL-17, TNF-alpha. IFN-gamma + IL-2 + IL-12/-18 seems to be the best combination.

Экспериментальное обоснование нового метода иммунодиагностики туберкулезных плевритов

The current study was perfomed to investigate the representativeness of the basic and antigen induced levels of specific (IFN-y) and non-specific (IL-6) biomarkers for the diagnosis of tuberculous pleural effusion. Ninety-three samples of pleural fluid from patient with tuberculosis (TP) and non-tuberculosis (Non-TP) pleurisy were examined. Induction of the synthesis IFN-gamma by immunocompetent cells in pleural exudate was carried out by the recombinant protein CFP10-ESAT6 contained in the diagnosticum «DIASKINTEST» by our method (Patent RF, № 2664427; 2017). Serum and pleural fluid IFN-y and IL-6 levels were measured by enzyme-linked immunosorbent assay technique (enzyme-am-plified sensitivity immunoassay kits, Vector-Best reagent kit (Russia). Our results suggested that introduction of M. tuberculosis antigens into the pleural fluids samples of TP group caused a significant increase of the IFN-y levels compared to basic level, which were not observed in the Non-TP group. Detection of IFN-gamma antigen-induced level in pleural fluid is a promising method for the diagnosis of tuberculous pleurisy. We found no significant differences in the basic and antigen-induced levels of IL-6 in patients with TP and Non-TP pleurisy.

Abstract 3746: Plasma membrane spanning and linker-domains from tumor necrosis factor receptor superfamily (TNFRSF) proteins provide novel functionality to chimeric antigen receptors (CARs) expressed in human T cells

Abstract Chimeric antigen receptor T cells redirected to the B cell antigen CD19 (CAR19) have shown efficacy in the treatment of B-lineage acute lymphocytic leukemia, and are being evaluated in other hematologic malignancies as well. The prevalent configuration of CAR19 for clinical application is comprised of the extracellular single chain targeting domain (scFv) derived from the FMC63 antibody, a CD8 linker and transmembrane domain, a CD137 signaling domain, and a CD3-zeta signaling domain. Transmembrane domains from other immunologically-active proteins, including CD3, CD28, or CD4, and spacer domains from IgG have been employed as structural components of CARs as well. The relationship between the composition of the CAR linker and transmembrane domain and CAR T function is, however, poorly understood. We hypothesized that CAR transmembrane and linker domains derived from proteins of the tumor necrosis factor receptor superfamily (TNFRSF), whose expression is associated with normal T cell functions, may optimize the anti-tumor activity of CAR19. Therefore, primary human T cells were transduced with lentiviral expression vectors (LV) expressing new combinations of linker and transmembrane sequences encoded by various TNFRSF members. Specifically, the linker:transmembrane domain combinations were: CD8:CD4, CD8:TNFRSF19, TNFRSF19:TNFRSF19, truncated TNFRSF19:TNFRSF19, TNFRSF9:TNFRSF9 and TNFRSF16:TNFRSF16. We found that CAR T cells comprised of transmembrane domains derived from either TNFRSF19 or TNFRSF9 were superior to the CD8 transmembrane CAR 19 in tumor killing, and in the induction of IFN gamma, TNF alpha, IL-2, and GM-CSF cytokine secretion in vitro, upon co-culture with leukemia cell line targets. Moreover, TNFRSF19-expressing CAR19 T cells were superior to the CD8 transmembrane CAR19 in the elimination of engrafted Raji cells (a Burkitt’s lymphoma line) in an NSG leukemia mouse model. On the other hand, the transmembrane domain derived from TNFRSF16/LNGFR, a low affinity neurotrophin receptor, created a CAR19 construct that had no functional activity either in vitro or in vivo, despite high levels of surface CAR expression in human T cells. In conclusion, the magnitude of CAR T response can be enhanced by the use of novel linker and transmembrane domains derived from TNFRSF19 and TNFRSF9. Thus, the transmembrane domain can play a determining role in CAR T function beyond a direct topographical connection between the extracellular and the intracellular domains of the chimeric protein. Studies are underway to elucidate the mechanistic role of TNFRSF-derived linker and transmembrane domains in CAR T function. Citation Format: Dina Schneider, Ying Xiong, Darong Wu, Boro Dropulic, Rimas Orentas. Plasma membrane spanning and linker-domains from tumor necrosis factor receptor superfamily (TNFRSF) proteins provide novel functionality to chimeric antigen receptors (CARs) expressed in human T cells [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 3746. doi:10.1158/1538-7445.AM2017-3746

Abstract 3678: Muc4 nanovaccine and checkpoint blockade based combination immunotherapy for pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is a highly metastatic and therapy-resistant malignancy characterized by immunosuppressive tumor microenvironment (TME) mucin overexpression. MUC4 is most differentially overexpressed mucin and functionally contributes to disease aggressiveness and is thus ideal candidate for targeted therapies. Tumor cells are also known to express programmed death ligand 1 (PD-L1) to suppress effector immune cells activity via PD-L1-PD-1 axis. We developed a novel vaccine using recombinant MUC4 fragments and exploiting adjuvant-like properties of amphiphilic polyanhydride-based nanoparticle delivery system. The strong involvement of MUC4 in disease aggressiveness and PD-L1 in immunosuppression makes a compelling case for their combined targeting. We hypothesize that combined overexpression of MUC4 and PD-L1 expression in pancreatic tumor microenvironment contributes to immunosuppressive and aggressive tumor behavior. Methods: Recombinant MUC4β fragment was encapsulated into polyanhydride nanoparticles (20:80 CPTEG:CPH) via nanoprecipitation. Murine PC cell lines (KCT960) derived from spontaneous pancreatic cancer mouse model (KPC mice) were transfected with human MUC4 expressing construct known as KCT960-mini-MUC4 cells for in-vivo studies. Flow cytometry, immunoblotting, PCR and immunofluorescence techniques were utilized to perform to characterize the immune response and target expression. Results: MUC4β nanovaccine immunized mice exhibited slower tumor growth kinetics than unimmunized control mice. We investigated tumor infiltrating lymphocytes (TILs) and necrosis in the tumor bed. We observed a positive correlation between TILs and tumor regression. Accumulation of infiltrating CD8+ and CD4+ T cells was greater in mice receiving the MUC4-Nanovaccine compared to soluble MUC4 delivered with blank nanoparticles, indicating the benefit of sustained availability of antigen via encapsulation. However, we did not observe complete tumor regression and ex vivo studies indicated induction of IFN-gamma by MUC4 vaccine. Based on this, we rationalized that PD-L1 expression by MUC4-expressing tumor cells suppressed and inhibited the therapeutic benefits of the nanovaccine in-vivo. We observed differential surface expression of PD-L1 on endogenously MUC4 expressing and negative PC cell lines. Paired analysis of MUC4 CRISPR knockdown and scrambled Capan-1 cell lines further suggested similar positive correlation. Conclusion: MUC4 nanovaccine suppressed tumor progression in-vivo indicating its potential for immunotherapy of PC. Positive correlation observed between MUC4 and PD-L1 in PC cell lines suggests an underlying molecular mechanism for establishing an immunosuppressive and aggressive PC tumor microenvironment and provides a strong rationale for evaluating MUC4 vaccine in combination with immune checkpoint blockade agents. Citation Format: Kasturi Banerjee, Shailendra Gautam, Waseem Mohd. Nasser, Prakash Kshirsagar, Kathleen Ross, Gaelle Spagnol, Abhijit Aithal, Paul Sorgen, Surinder Kumar Batra, Sushil Kumar, Balaji Narasimhan, Maneesh Jain. Muc4 nanovaccine and checkpoint blockade based combination immunotherapy for pancreatic cancer [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 3678. doi:10.1158/1538-7445.AM2017-3678

Abstract 4138: In vitro and in vivo models for evaluation of inhibitors of tryptophan dioxygenases

Abstract Cancers co-opt indoleamine 2,3-dioxygenase 1 (IDO1) as a mechanism for evading the immune system, and IDO1 inhibitors have emerged as a promising new approach for the treatment cancer through their potential to restore antitumor immunity and to synergise with existing therapies. As part of our investigations into novel small molecule inhibitors of IDO1, we screened a number of human and murine tumor lines for expression of tryptophan dioxygenases. All the human and murine tumor lines that we tested were found not to express tryptophan dioxygenases unless induced with IFN-gamma. Our screen included testing 50 primary human melanoma lines developed from tumor samples obtained New Zealand Melanoma patients (NZMel lines) for expression of IDO1 and IDO2. After co-culture with IFN-gamma, 84% of the NZMel lines were IDO1+ and IDO2+; 4% were IDO1+ only; 2 % were IDO2+ only; whilst 10% did not express either IDO1 or IDO2. Tryprophan dioxygenase (TDO) was not detected in any of the NZMel lines before or after IFN-gamma exposure. To overcome the need for IFN-gamma induction for our cell based assays of IDO1 inhibitory activity of our novel compounds, we transfected the wild-type murine Lewis Lung carcinoma line (LLTC) to constitutively express murine IDO1 (LLTC-mIDO1) or human IDO1 (LLTC-hIDO1), and used these engineered lines to assay for potential species selectivity of our inhibitors. IDO1 expression in the lines was consistent and stable, but when LLTC-hIDO1 cells were implanted subcutaneously into syngeneic mice for in vivo testing of inhibitors, we found considerable heterogeneity in IDO1 expression between tumors. Approximately half of the tumors from the same batch of implants were completely negative for IDO1 expression by Western blot analysis. More intriguingly, when fragments of an IDO1+ or an IDO1- donor tumor were implanted into new recipients, the same degree of intertumoral heterogeneity in IDO1 expression was seen in the subsequent generation of tumors. In contrast, subcutaneous tumors developing from murine GL261 glioma cells similarly engineered to over-express hIDO1 (GL261-hIDO1) were homogeneous in their expression of hIDO1, and were used for subsequent in vivo studies of intratumoral IDO1expression on its effects on immune cell infiltrates and tumor growth. GL261-hIDO1 tumors had a significantly faster growth rate than the wild-type tumors, and 24 days after implantation, had reached a mean tumor volume of 3600 mm3 compared to a mean tumor volume of 500 mm3 of wild-type tumors. Analysis of the immune cell infiltrates, showed that 14-day GL261 wild-type tumors had a 3-fold higher percentage of CD3+ T cells than that in GL261-hIDO1 tumors. CD8+ cells made up 55% and 25% of the CD3+ cells in wild-type and GL261-hIDO1 tumors, respectively. Percentage of Foxp3+ (Treg) cells was higher in GL261-hIDO1 tumors compared to that in wild-type tumors. Citation Format: Lai-Ming Ching, Petr Tomek, Brian D. Palmer, Sofian Tijono, Kimiora Henare, Lukas M. Braun. In vitro and in vivo models for evaluation of inhibitors of tryptophan dioxygenases [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 4138. doi:10.1158/1538-7445.AM2017-4138

Effect of exogenous interferon-gamma (IFN-gamma) on peripheral blood immune markers as part of a phase I clinical trial of combined IFN-gamma with nivolumab (Nivo) in patients (pts) with select solid tumors.

97 Background: IFN-gamma is a known activator of PD-L1 expression and plays a key role in immune activation. We present initial correlative peripheral blood findings after the first 2 cohorts of pts enrolled in a phase I trial of combined IFN-gamma/Nivo in pts with various solid tumors. Methods: Pts with advanced solid tumors who had progressed after at least 1 prior therapy were recruited. An induction phase of IFN-gamma (50 or 75 mcg/m2 subq every other day for 1 week) preceded the combination of Nivo (3 mg/kg IV every 2 weeks) with continuation of IFN-gamma at same dose and schedule for 3 months. Pts with clinical benefit could remain on Nivo alone for up to 1 year. Peripheral blood was analyzed via flow cytometry at baseline, after IFN-gamma induction (prior to Nivo start), and after 6 weeks of combo treatment. Results: 13 pts have been accrued. Pre and post induction samples were collected on all pts. Nine pts had available on-treatment samples at time of data analysis. Comparing blood collections from baseline to post-IFN-gamma induction revealed a substantial expansion of the CD14high, CD16+ monocyte population (p=0.001) and a statistically significant increase in MHC class II expression on all monocyte populations (p≤ 0.002 for all types). Natural killer (NK) cells demonstrated greater numbers of cells per ml of blood (p=0.03) and increased NKp30 expression, an important receptor mediating tumor cell lysis (p= 0.005 for CD56dim; p= 0.03 for CD56bright). With the addition of Nivo, evidence for activation of CD56dim NK cells was detected as increased CD69 expression compared to post-induction (p=0.02). PD-1 expression on T-cells showed reduced expression in most comparators after initiation of Nivo (p=0.055 CD8+; p=0.008 CD4+). Conclusions: Early correlative peripheral blood data demonstrate expected systemic IFN-gamma effect as measured by monocyte activation and increased MHC II expression. Evidence of NK cell activation and proliferation was also observed. Decreased PD-1 expression on T-cells may reflect down-regulation in response to PD-1 blockade. Correlations with biopsy specimens and clinical response are planned. Clinical trial information: NCT02614456.

Abstract A75: Meloxicam and nimesulide modulate immunity in pancreatic cancer

Abstract Background: Meloxicam and nimesulide have been studied as potential cancer immunomodulatory agents. Those drugs have been shown to have immune stimulatory effects on the tumor stroma and the microenvironment of several tumors including pancreatic ductal adenocarcinoma. They have several immune effects in vitro and in animal models. We compared the immunomodulatory activity of two preferential Cox-2 inhibitors drugs, meloxicam and nimesulide in advanced pancreatic cancer patient’s peripheral blood mononuclear cells (PBMCs) with good and poor Karnofsky. Materials and Methods: Repository PBMCs from advanced pancreatic patients with Karnofsky < 50% (n=10) and > 80% (n=10) were stimulated with different concentrations of meloxicam and nimesulide in vitro. We performed dose-response curve with concentrations of both drugs ranging from 0.5, 1, 10, 50 and 100 μM and we performed the ELISPOT assays for Interferon-gamma using human anti-CD3 and CMV whole lysate as basal T cell stimulation. Th2 cytokines were measured as well by ELISPOT and Cytokine ELISA. Results: In patients with Karnofsky > 80% both meloxicam and nimesulide were effective in promoting IFN-gamma production after stimulation with both drugs starting at 0.5 μM. The use of meloxicam was associated with significant production of IFN-gamma at 0.5 μM (p<0.001) and 1μM (p<0.0001). Nimesulide significantly induced IFN-gamma secretion at 0.5 μM (p=0.005), 1 μM (p=0.0002) and at 10 μM (p<0.0001). Meloxicam and nimesulide treatment resulted in different responses in PBMCs IFG-gamma production from pancreatic cancer patients that had a Karnofsky <50%. Nimesulide was more effective than meloxicam at promoting IFN-gamma production at 1μM (p<0.005) and 10μM (p<0.0001). Meloxicam did not improve the IFN-gamma secretion at low doses such as 0.5, 1 and 10 μM (n.s.). However, meloxicam at 50 μM and 100 μM was able to induce IFN-gamma (p<0.05 and p<0.0001, respectively). Additionally PBMCs treated with nimesulide in both groups were able to decrease Th2 cytokines such as IL-6, IL-18, TNF-alpha and IL-8 (statistical analysis in progress). Discussion: This preliminary data suggests that the use of meloxicam and nimesulide as putative immunomodulatory drugs may be less effective when pancreatic patients have a poor Karnosky although at high concentration they are effective. Also, the potent induction of IFN-gamma in patients with a good Karnofsky suggest synergy of these drugs in combination with standard of care treatment, immunogenic chemotherapy, anti-stroma strategies and active specific immunotherapy as a combinatorial approach to move forward into animal models of pancreatic cancer and eventually into the clinic. Grant Support: Junior Physician Scientist Fellowship Grant from CICS. Citation Format: Juan Marquez, Erik Ramos, Pedro Alejandro Lucero-Diaz, Fernando Durazo-Bustamante, Gerardo Palacios Diaz De Leon, Rosana Soto-Soto, Sara Carrillo-Marquez, Arturo Suplee-Rivera, Alejandro Camacho-Hernandez, Daniel Camacho-Gil.{Authors}. Meloxicam and nimesulide modulate immunity in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A75.

Abstract 2297: Tandem anti-CD20 and -CD19 scFV-based chimeric antigen receptors (CARs) mitigate tumor escape in hematologic malignancies

Abstract In recent years, adoptive cell therapy using T cells engineered with chimeric antigen receptors (CAR T) specific for CD19 has shown marked clinical efficacy. Nevertheless, cases of tumor escape due to the loss of CD19 antigen have been reported. We hypothesized that tumor escape in B cell malignancies could be mitigated by CAR T approaches that target the CD19 and CD20 tumor antigens simultaneously. Second generation CARs containing scFv specific for CD19 and CD20 were generated by lentiviral transduction of human primary T cells. The configurations were: a) single targeting domain, b) tandem targeting domains, or c) two full-length CAR receptors co-expressed bicistronically in the same T cell. CAR T expression for all constructs was confirmed by Western blotting and protein L flow cytometry. CAR T cytolytic activity and IFN gamma secretion were evaluated by killing assays and ELISA, respectively. Single, tandem, and bicistronic CAR T cells exhibited CAR surface expression of 50-70%. All anti-CD19 and anti-CD20 CAR T cells demonstrated target-specific cells lysis and induction of IFN gamma. Interestingly, the bicistronic CAR19 CAR20 construct yielded high CAR T surface expression and IFN gamma production, but inferior in vitro cytolytic activity. To confirm CAR T specificity, K562 CD19+ and K562 CD20+ cell lines were generated. In flow-based 1h killing assays, CAR19 and CAR20 lysed their respective target lines only, whereas tandem CAR constructs lysed both K562 CD19+ and K562 CD20+ cells, but not K562 control. In a subsequent series of co-culture experiments, Raji target cells were combined with very low ratios of CAR T cells, to allow for the evolution of escape variants. On day 5 of co-incubation in the presence of CAR19 T cells, the surviving Raji cells numbers were comparable to control (85,000 cells/well vs 77, 000 cells/well in sham control). In comparison, viable Raji numbers decreased drastically post co-incubation with CAR20-, CAR19_20- and CAR20_19-T cells (546, 1,355 and 1,543 cells/well, respectively). Among the surviving Raji population, CD19 surface expression was reduced to 1.6%, vs 93.29% for sham control, whereas the expression of CD20 and CD22 remained high (97% and 78%, respectively). By contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. Thus, targeting two tumor antigens simultaneously using tandem CAR T cells is a promising new strategy for mitigation of tumor escape via antigen down-regulation. Moreover, expression of two scFv in a single CAR format is superior to expression of two independent CARs from a bicitsronic vector. It also appears that the order of the targeting domains within the tandem CAR impacts anti-tumor activity. These findings are currently being explored further in animal model systems. Citation Format: Dina Schneider, Ying Xiong, Darong Wu, Boro Dropulic, Rimas Orentas. Tandem anti-CD20 and -CD19 scFV-based chimeric antigen receptors (CARs) mitigate tumor escape in hematologic malignancies. [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 2297.

648. Mitigating Tumor Escape: Tandem Anti-CD20- and CD19 SCFV-Based Chimeric Antigen Receptors (CARs) in Leukemia/Lymphoma

In recent years, adoptive cell therapy using T cells engineered with chimeric antigen receptors (CAR T) specific for CD19 has shown marked clinical efficacy. Nevertheless, cases of tumor escape due to tumor escape in B cell malignancies could be mitigated by CAR T the loss of CD19 antigen have been reported. We hypothesized that approaches that target the CD19 and CD20 tumor antigens simultaneously. Second generation CARs containing scFv specific for CD19 and CD20 were generated by lentiviral transduction of human primary T cells. The configurations were: a) single targeting domain, b) tandem targeting domains, or c) two full-length CAR receptors co-expressed bicistronically in the same T cell. CAR T expression for all constructs confirmed by Western blotting and protein L flow cytometry. CAR T cytolytic activity and IFN gamma secretion were evaluated by killing assays and ELISA, respectively. Single, tandem, and bicistronic CAR T cells exhibited CAR surface expression of 50-70%. All anti- CD19, anti-CD20 CAR T cells demonstrated target-specific cells lysis and induction of IFN-gamma. Interestingly, bicistronic CAR19, CAR20 construct yielded high CAR T surface expression and IFN-gamma production, but inferior in vitro cytolytic activity. To confirm CAR T specificity, K562 CD19+ and K562 CD20+ cell lines were generated. In flow-based 1h killing assays, CAR19 and CAR20 lysed their respective target lines only, whereas tandem CAR constructs lysed both K562 CD19+ and K562 CD20+ cells, but not K562 control. In a subsequent series of co-culture experiments, Raji target cells were combined with very low ratios of CAR T cells, to allow for the evolution of escape variants. On day 5 of co-incubation in the presence of CAR19 T cells, the surviving Raji cells numbers were comparable to control (85,000 cells/well vs 77, 000 cells/well in sham transduction). In comparison, viable Raji numbers decreased drastically post co-incubation with CAR20-, CAR19_20- and CAR20_19-T cells (546, 1,355 and 1,543 cells/well, respectively). Among the surviving Raji population, CD19 surface expression was reduced to 1.6%, vs 93.29% for sham control, whereas the expression of CD20 and CD22 remained high (97% and 78%, respectively). By contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. Thus, targeting two tumor antigens simultaneously using tandem CAR T cells is a promising new strategy for mitigation of tumor escape via antigen down-regulation. Moreover, expression of two scFv in a single CAR format is superior to expression of two independent CARs from a bicitsronic vector. It also appears that the order of the targeting domains within the tandem CAR impacts anti-tumor activity. These findings are currently being explored further in animal model systems.

Interleukin 18 (IL-18) as a target for immune intervention.

Interleukin 18 (IL-18) is a pleiotropic cytokine involved in the regulation of innate and acquired immune response. In the milieu of IL-12 or IL-15, IL-18 is a potent inducer of IFN-gamma in natural killer (NK) cells and CD4 T helper (Th) 1 lymphocytes. However, IL-18 also modulates Th2 and Th17 cell responses, as well as the activity of CD8 cytotoxic cells and neutrophils, in a host microenvironment-dependent manner. It is produced by various hematopoietic and nonhematopoietic cells, including dendritic cells and macrophages. In an organism, bioactivity of the cytokine depends on the intensity of IL-18 production, the level of its natural inhibitory protein - IL-18BP (IL-18 binding protein) and the surface expression of IL-18 receptors (IL-18R) on the responding cells. This review summarizes the biology of the IL-18/IL-18BP/IL-18R system and its role in the host defense against infections. The prospects for IL-18 application in immunotherapeutic or prophylactic interventions in infectious and non-infectious diseases are discussed.

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Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.

Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection.

The response to multi-drug resistant bacterial infections must be a global priority. While mounting resistance threatens to create what the World Health Organization has termed a “post-antibiotic era”, the recent discovery that antibiotic use may adversely impact the microbiome adds further urgency to the need for new developmental approaches for anti-pathogen treatments. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, has declared itself a serious threat within the United States and abroad. A potential solution to the problem of antibiotic resistance may not entail looking to the future for completely novel treatments, but instead looking into our history of bacteriophage therapy. This study aimed to test the efficacy, safety, and commercial viability of the use of phages to treat Staphylococcus aureus infections using the commercially available phage SATA-8505. We found that SATA-8505 effectively controls S. aureus growth and reduces bacterial viability both in vitro and in a skin infection mouse model. However, this killing effect was not observed when phage was cultured in the presence of human whole blood. SATA-8505 did not induce inflammatory responses in peripheral blood mononuclear cultures. However, phage did induce IFN gamma production in primary human keratinocyte cultures and induced inflammatory responses in our mouse models, particularly in a mouse model of chronic granulomatous disease. Our findings support the potential efficacy of phage therapy, although regulatory and market factors may limit its wider investigation and use.

Role of Baicalin in Anti-Influenza Virus A as a Potent Inducer of IFN-Gamma.

Baicalin (BA) is a flavonoid compound purified from Scutellaria baicalensis Georgi and has been shown to possess a potent inhibitory activity against viruses. However, the role of BA in anti-influenza virus has not been extensively studied, and the immunological mechanism of BA in antiviral activity remains unknown. Here, we observed that BA could protect mice from infection by influenza virus A/PR/8/34 (H1N1), associated with increasing IFN-γ production, but presented no effects in IFN-γ or IFN-γ receptor deficient mice. Further study indicated that BA could inhibit A/PR/8/34 replication through IFN-γ in human PBMC. Moreover, BA can directly induce IFN-γ production in human CD4+ and CD8+ T cells and NK cells, and activate JAK/STAT-1 signaling pathway. Collectively, BA exhibited anti-influenza virus A (H1N1) activity in vitro and in vivo as a potent inducer of IFN-γ in major IFN-γ producing cells.

Rapid screening of healthy donors suggests strategies for clinical purification of virus-specific T cells

Transfer of virus-specific memory T cells is an emerging treatment for viral diseases in immunocompromised patients. One approach is to purify antigen-specific cells based on their up-regulation of activation markers after short-term in vitro stimulation with peptides. In order to rapidly screen the frequency of antigenspecific CD4 and CD8 T cells in potential donors we have modified a commercial kit fromMiltenyi so that we can simultaneouslymeasure induction of IFN-gamma, CD40L and CD137 on both cell types following peptide stimulation. In a prestudy using blood from healthy donors we found that the presence of IFN-gamma producing cells following stimulation with EBV or CMV peptides correlated with the presence of serum antibodies against the viruses. Simultaneous stimulationwith peptides derived frommultiple proteins lead to activation of similar number of cells as stimulation with separate peptides. Whereas it was relatively easy to identify memory cells reactive to CMV and EBV, fewer donors maintained cells that could be activated by peptides derived from JC and BK viruses. IFN-gamma producing cells had increased expression of CD137, and CD4 cells also CD40L. However, more cells up-regulated their expression of CD137 than IFN-gamma following peptide stimulation and background levels of CD137 expression was higher, demonstrating that care has to be taken when selecting activation marker for purification. Taken together, this pre-study to clinical purification of virus-specific memory T cells shows that rapid screening of potential donors is possible, and suggests strategies for activation and purification of virus-specific cells.

Induction of interferon-gamma and downstream pathways during establishment of fetal persistent infection with bovine viral diarrhea virus

Development of transplacental infection depends on the ability of the virus to cross the placenta and replicate within the fetus while counteracting maternal and fetal immune responses. Unfortunately, little is known about this complex process. Non-cytopathic (ncp) strains of bovine viral diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, cause persistent infection in early gestational fetuses (<150 days; persistently infected, PI), but are cleared by immunocompetent animals and late gestational fetuses (>150 days; transiently infected, TI). Evasion of innate immune response and development of immunotolerance to ncp BVDV have been suggested as possible mechanisms for the establishment of the persistent infection. Previously we have observed a robust temporal induction of interferon (IFN) type I (innate immune response) and upregulation of IFN stimulated genes (ISGs) in BVDV TI fetuses. Modest chronic upregulation of ISGs in PI fetuses and calves reflects a stimulated innate immune response during persistent BVDV infection. We hypothesized that establishing persistent fetal BVDV infection is also accompanied by the induction of IFN-gamma (IFN-γ). The aims of the present study were to determine IFN-γ concentration in blood and amniotic fluid from control, TI and PI fetuses during BVDV infection and analyze induction of the IFN-γ downstream pathways in fetal lymphoid tissues. Two experiments with in vivo BVDV infections were completed. In Experiment 1, pregnant heifers were infected with ncp BVDV type 2 on day 75 or 175 of gestation or kept naïve to generate PI, TI and control fetuses, respectively. Fetuses were collected by Cesarean section on day 190. In Experiment 2, fetuses were collected on days 82, 89, 97, 192 and 245 following infection of pregnant heifers on day 75 of gestation. The results were consistent with the hypothesis that ncp BVDV infection induces IFN-γ secretion during acute infection in both TI and PI fetuses and that lymphoid tissues such as spleen, liver and thymus, serve both as possible sources of IFN-γ and target organs for its effects. Notably, induction of IFN-γ coincides with a decrease in BVDV RNA concentrations in PI fetal blood and tissues. This is the first report indicating the possible presence of an adaptive immune response in persistent BVDV infections, which may be contributing to the observed reduction of viremia in PI fetuses.