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Introduction: The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells. Induction of PD-L1 expression on cancer cells is associated with inhibition of immune responses against cancer, thus favouring cancer progression and metastasis. Activation of PD-1/PD-L1 pathway induces T-cell anergy and exhaustion and enhances the function of regulatory T-cells (Tregs) thereby leading to an immune suppressive environment. Therefore, blocking this pathway restores the proliferation and cytotoxicity of T- lymphocytes, inhibits the function of Tregs and results in decreased T-cell apoptosis. A number of monoclonal antibodies agents targeting PD-1/PD-L1 have approved for a number of malignancies. These approved therapies require bolus intravenous injections, are administered in high dose and have a long half-life. The long residence time of these mAbs could contribute to the well-documented drug-related adverse effects. Therefore, there is still a need for potent, selective small molecule inhibitors of the PD-1/PD-L1 pathway. Such small molecule inhibitors, can provide increased oral bioavailability, and shorter half-life activity for a more controllable treatment, and flexibility for combination strategies. Methods: Rational design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signaling reporter assays as well as ex-vivo co-culture assays were used to assess the ability of the compounds to restore T-cell proliferation and function. In vivo efficacy was assessed by syngeneic and humanized models in mice. Results: Our lead molecule JTi showed strong in vitro IC50 of 0.8 nM in TR-FRET assay that measures interaction between PD-1 and PD-L1. This molecule also augmented T-cell response as measured by IFN-gamma activation in a cancer cell-PBMC based co-culture assay. This molecule induced dimerization of PD-L1 in U2OS cell based assay with an EC50 of ~300 nM. Competition study between anti-PD-L1 blocking antibody and this small molecule inhibitor clearly suggested that it binds at the same site as the antibody. In a cell based assay, measuring PD-1/PD-L1 interaction, JTi clearly demonstrated inhibition of PD-1 and PD-L1 binding in a dose-dependent manner. JTi showed comparable efficacy to the anti-PD-L1 antibody in syngeneic (4T1, CT-26) and humanized models (MC-38/hPD-L1) by oral administration. Conclusion: Advanced studies to further characterize and progress the molecule into pre-clinical development are in progress. The oral administration route of these small molecule PD-1/PD-L1 inhibitors would provide an attractive option to be used as a maintenance therapy followed by mAb based treatment to enhance patient compliance. The lead compound will be entering IND enabling studies in the 1st half of 2021. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Chandregowda venkateshappa, Muralidhar Reddy, Pendyala Satya Kishore, Pratima Deshpande, Sundarajan kannan, Tabassum Sahareen, Santhosh Viswakarma, Amir Siddiqui, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Rashmi Rekha Devi. Novel, small molecule inhibitors of PD-L1/PD-1 interaction [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 1630.

Security in Embedded systems has become an important constraint in modern days. There are different cryptographic algorithms developed for providing data security. Among these Diffie-Hellman is the basic one. Diffie-Hellman Key Exchange method of cryptography has been the simplest method of implementation used in various cryptographic applications till now. In this work, we introduce the Diffie-Hellman based cryptography implementation on two embedded devices communicating with each other over Ethernet. Our aim is to provide a secure data communication by using key agreement scheme generated using the Diffie-Hellman key exchange algorithm Here, we are implementing the Diffie-Hellman based cryptography on Raspberry Pi and arduino hardware by establishing a socket communication between the two hardware devices and the key exchange happens using the established network communication.

Immune responses to citrullinated neoantigens and clinical efficacy of costimulation blockade indicate a general defect in maintaining T cell tolerance in rheumatoid arthritis (RA). To examine whether TCR threshold calibration contributes to disease pathogenesis, signaling in RA T cells was quantified. RA patients had a selective increase in ERK phosphorylation compared with demographically matched controls due to a mechanism distal of Ras activation. Increased ERK responses included naive and memory CD4 and CD8 T cells and did not correlate with disease activity. The augmented ERK activity delayed SHP-1 recruitment to the TCR synapse and sustained TCR-induced Zap70 and NF-κB signaling, facilitating responses to suboptimal stimulation. Increased responsiveness of the ERK pathway was also a characteristic finding in the SKG mouse model of RA where it preceded clinical symptoms. Treatment with subtherapeutic doses of a MEK-1/2 inhibitor delayed arthritis onset and reduced severity, suggesting that increased ERK phosphorylation predisposes for autoimmunity and can be targeted to prevent disease.

Background: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine-based inhibitors including JQ1, I-BET762, and OTX015. Phase I clinical trials have now been initiated with this agent based on its potent anti-tumor activity in various in vitro and in vivo models of hematologic malignancies and solid tumors. In view of the recent publications implicating a role for BET protein BRD4 in the suppression of PD-L1 expression, an immune checkpoint ligand for PD-1, we sought to evaluate ODM-207 for its effect on immune-mediated anti-tumor efficacy in pre-clinical models. Methods and Results: Mouse splenocytes were stimulated with anti-CD3 and anti-CD28 in the presence or absence of ODM-207 for four days and changes in immune cell population were analyzed by FACS. Results revealed an increase in the level of activated cytotoxic CD8+ T cells as indicated by increased intracellular IFNγ and granzyme B with ODM-207 treatment. After confirming the lack of direct anti-proliferative activity on the mouse colon carcinoma cell line CT26, in vivo evaluation of ODM-207 was carried out in the syngeneic CT26 subcutaneous tumor model established in BALB/c mice. Daily oral administration of ODM-207 at 30 mg/kg was well tolerated in this model and resulted in a statistically significant inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ODM-207 was comparable to that with a commercially available anti-mouse PD1 antibody. Studies to characterize the immune changes in the tumor and anti-tumor activity of ODM-207 in combination with an anti-mouse PD1 antibody are currently underway and the results will be presented. Conclusions: In summary, these studies demonstrate the anti-tumor activity of BET inhibitor in a syngeneic model of colon carcinoma in the absence of a direct anti-proliferative activity on tumor cells. Observed tumor growth inhibition correlated with the in vitro activation of cytotoxic CD8+ T cells supporting the immune-mediated effect leading to tumor growth inhibition. In view of the remarkable success with the immune-based therapeutic approaches, these findings are relevant in devising appropriate strategies for the continued clinical development of ODM-207. Citation Format: Pratima Deshpande, Ravi Krishna Babu, Prashant Yallappa Vadnal, Mahaboobi Jaleel, Murali Ramachandra, Chandrasekhar Abbineni, Susanta Samajdar, Anu-Maarit Moilanen, Pekka Kallio. Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models [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 LB-113. doi:10.1158/1538-7445.AM2017-LB-113

Regulation of the ERK pathway is intimately involved in determining whether TCR stimulation is productive or induces anergy. T cells from patients with rheumatoid arthritis (RA) have increased ERK responsiveness, which may be relevant for disease pathogenesis. Inflammatory cytokines such as TNF-α did not reproduce the TCR hypersensitivity typical for RA in T cells from healthy individuals. In contrast, priming with the homeostatic cytokines (HCs) IL-7 and IL-15 amplified ERK phosphorylation to TCR stimulation 2- to 3-fold. The underlying mechanism involved a priming of the SOS-dependent amplification loop of RAS activation. The sensitization of the TCR signaling pathway has downstream consequences, such as increased proliferation and preferential Th1 differentiation. Importantly, priming with IL-7 or IL-15 enabled T cell responses to autoantigens associated with RA. Production of HCs is induced in lymphopenic conditions, which have been shown to predispose for autoimmunity and which appear to be present in the preclinical stages of RA. We propose that HCs, possibly induced by lymphopenia, decrease the signaling threshold for TCR activation and are thereby partly responsible for autoimmunity in RA.

Autoantibodies to common autoantigens and neoantigens, such as IgG Fc and citrullinated peptides, are immunological hallmarks of rheumatoid arthritis (RA). We examined whether a failure in maintaining tolerance is mediated by defects in T-cell receptor activation threshold settings. RA T cells responded to stimulation with significantly higher ERK phosphorylation ( P < 0.001). Gene expression arrays of ERK pathway members suggested a higher expression of KRAS and BRAF , which was confirmed by quantitative PCR ( P = 0.003), Western blot, and flow cytometry ( P < 0.01). Partial silencing of KRAS and BRAF lowered activation-induced phosphorylated ERK levels ( P < 0.01). In individual cells, levels of these signaling molecules correlated with ERK phosphorylation, attesting that their concentrations are functionally important. In confocal studies, B-RAF/K-RAS clustering was increased in RA T cells 2 min after T-cell receptor stimulation ( P < 0.001). Overexpression of B-RAF and K-RAS in normal CD4 T cells amplified polyclonal T-cell proliferation and facilitated responses to citrullinated peptides. We propose that increased expression of B-RAF and K-RAS lowers T-cell activation thresholds in RA T cells, enabling responses to autoantigens.

CD11c+ dendritic cells (DCs) are a prominent component of CNS infiltrates in mice with experimental autoimmune encephalomyelitis. However, their role in immunopathogenesis is controversial. In this study, we report that they originate from peripheral hemopoietic cells and exhibit diverse functions that change during the course of acute disease. CNS DCs stimulate naive T cells to proliferate and polarize Th17 responses when harvested shortly following disease onset but are relatively inefficient APC by the time of peak disability. Conversely, they can support CD4+CD25+ T cell-mediated immunosuppression early during experimental autoimmune encephalomyelitis. Such paradoxical functions might reflect dual roles of CNS DCs in promoting local inflammation while setting the stage for remission.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system. IL-12p40 monokines play a critical role in the generation of EAE-inducing CD4+T cells. Here we show that IL-12 directly upregulates the expression of the adhesion molecule, P-selectin glycoprotein ligand (PSGL-1), on B10.PL MBP-TCR transgenic T cells during their initial encounter with antigen. Pre-incubation of IL-12-stimulated myelin-reactive CD4+T cells with a blocking antibody against PSGL-1 reduced the incidence and severity of EAE. We conclude that IL-12-driven PSGL-1 expression can facilitate the development of autoimmune demyelination.

In animal models, lymphopenia and the associated function of homeostatic cytokines (HC) set the stage for autoimmunity. This model is relevant for rheumatoid arthritis (RA) which has defects in T cell homeostasis due to a failure in DNA repair and which responds to JAK inhibition. We have previously shown that RA T cells have a hyperreactive ERK response which impairs the induction of anergy. Here, we examined whether HC tune TCR activation thresholds by calibrating the ERK rheostat. RA T cells exhibited increased levels of phosphorylated STAT3 and STAT5 indicating in vivo activity of IL-21/IL6 and IL-7/IL-15. Incubation of healthy donor T cells with IL-7, IL15 or IL21 prior to anti-CD3/CD28 stimulation augmented ERK activation and expression of activation markers (CD69, CD40L) and cytokines (TNFα, IFNγ and IL-17). Preincubation with IL-1β or TNFα did not exert an effect. In DR4 healthy donors, HC priming facilitated responses to self-antigens such as citrullinated vimentin peptide. The effect of HC lasted for less than 3 hours and was sensitive to PI3K inhibition suggesting a non-transcriptional PI3K mediated priming of the ERK pathway. HC dose titration showed a bimodal distribution in pERK consistent with an on-off switch characteristic of a positive feedback loop. Allosteric binding of RAS-GTP to SOS is known to induce such an on-off switch. Indeed, HC incubation increased active RAS. We propose that HC lower the threshold for autoimmune responses by priming of SOS.

Immune responses to citrullinated neoantigens indicate a tolerance defect in rheumatoid arthritis (RA). We examined the hypothesis that abnormal TCR signaling in RA T cells lowers the activation threshold. T cells from 33 seropositive RA patients and 33 controls were stimulated by CD3/CD28 crosslinking or superantigen-coated dendritic cells. Activation-induced signaling was quantified by PhosFlow, Western blotting, and confocal microscopy. Basal as well as post-stimulation p-ERK was higher in naïve, memory and effector CD4 and CD8 T cells from RA patients compared to controls (p