Process Development of a Macrocyclic Peptide Inhibitor of PD-L1.

This article outlines the process development leading to the manufacture of 800 g of BMS-986189, a macrocyclic peptide active pharmaceutical ingredient. Multiple N-methylated unnatural amino acids posed challenges to manufacturing due to the lability of the peptide to cleavage during global side chain deprotection and precipitation steps. These issues were exacerbated upon scale-up, resulting in severe yield loss and necessitating careful impurity identification, understanding the root cause of impurity formation, and process optimization to deliver a scalable synthesis. A systematic study of macrocyclization with its dependence on concentration and pH is presented. In addition, a side chain protected peptide synthesis is discussed where the macrocyclic protected peptide is extremely labile to hydrolysis. A computational study explains the root cause of the increased lability of macrocyclic peptide over linear peptide to hydrolysis. A process solution involving the use of labile protecting groups is discussed. Overall, the article highlights the advancements achieved to enable scalable synthesis of an unusually labile macrocyclic peptide by solid-phase peptide synthesis. The sustainability metric indicates the final preparative chromatography drives a significant fraction of a high process mass intensity (PMI).

Plasma sPD-L1 and VEGF levels are associated with the prognosis of NSCLC patients treated with combination immunotherapy.

The clinical significance of plasma soluble programmed cell death ligand 1 (sPD-L1) and vascular endothelial growth factor (VEGF) for non-small cell lung cancer (NSCLC) treated with the combination of anti-angiogenic therapy and anti-PD-L1 antibody (Ab) remain unknown. This study aimed to explore the association between plasma sPD-L1 and VEGF levels and the prognosis of NSCLC patients treated with the combination of Envafolimab and Endostar. Peripheral blood samples were collected from 24 NSCLC patients at baseline and after 6 weeks of treatment and were detected for sPD-L1 and VEGF levels. Both baseline and posttreatment sPD-L1 were significantly higher in progressive disease (PD) group than in controlled disease (CD) group (median: 77.5 pg/ml vs. 64.6 pg/ml, P  = 0.036, median: 8451 pg/ml vs. 5563 pg/ml, P  = 0.012). In multivariate analysis, lower baseline sPD-L1 levels were significantly associated with longer progression-free survival (PFS) (HR = 6.834, 95% CI: 1.350-34.592, P  = 0.020). There were significantly higher posttreatment VEGF levels in PD group compared with CD group (median: 323.7 pg/ml vs. 178.5 pg/ml, P  = 0.009). Higher posttreatment VEGF levels were significantly associated with shorter PFS in multivariate analysis (HR = 5.911, 95% CI: 1.391-25.122, P  = 0.016). Plasma sPD-L1 and VEGF levels are associated with the clinical response and prognosis of NSCLC patients treated with the combination of PD-L1 inhibitors and anti-angiogenetic therapy.

Structure-Guided Discovery of PD-1/PD-L1 Interaction Inhibitors: Peptide Design, Screening, and Optimization via Computation-Aided Phage Display Engineering.

Cancer immunotherapy harnesses the immune system to combat tumors and has emerged as a major cancer treatment modality. The PD-1/PD-L1 immune checkpoint modulates interactions between tumor cells and T cells and has been extensively targeted in cancer immunotherapy. However, the monoclonal antibodies known to target this immune checkpoint have considerable side effects, and novel PD-1/PD-L1 inhibitors are therefore required. Herein, a peptide inhibitor to disrupt PD-1/PD-L1 interactions was designed through structure-driven phage display engineering coupled to computational modification and optimization. BetaPb, a novel peptide library constructed by using the known structure of PD-1/PD-L, was used to develop inhibitors against the immune checkpoint, and specific peptides with high affinity toward PD-1 were screened through enzyme-linked immunosorbent assays, homogeneous time-resolved fluorescence, and biolayer interferometry. A potential inhibitor, B8, was preliminarily screened through biopanning. The binding affinity of B8 toward PD-1 was confirmed through computation-aided optimization. Assessment of B8 variants (B8.1, B8.2, B8.3, B8.4, and B8.5) demonstrated their attenuation of PD-1/PD-L1 interactions. B8.4 exhibited the strongest attenuation efficiency at a half-maximal effective concentration of 0.1 µM and the strongest binding affinity to PD-1 (equilibrium dissociation constant = 0.1 µM). B8.4 outperformed the known PD-1/PD-L1 interaction inhibitor PL120131 in disrupting PD-1/PD-L1 interactions, revealing that B8.4 has remarkable potential for modification to yield an antitumor agent. This study provides valuable information for the future development of peptide-based drugs, therapeutics, and immunotherapies for cancer.

The detection of PD-L1 expression on liquid-based cytology in pleural effusion of lung adenocarcinoma and its prognostic evaluation: Between paired liquid-based cytology and cell block samples.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression levels measured by immunohistochemistry have been proven to predict the outcome of immunotherapy in lung adenocarcinoma (LUAD). However, data on PD-L1 expression on liquid-based cytology (LBC) in malignant pleural effusion (MPE) is scarce. METHODS: This study cohort included 60 cases with MPE suffering from LUAD. PD-L1 SP263 assay was used for immunocytochemistry (ICC) on LBC and matched cell block (CB) to validate ICC protocols on LBC slides. Clinical outcomes were analyzed based on immunotherapy and PD-L1 tumor proportion scores (TPS) on LBC slides and CBs. RESULTS: PD-L1 expression with TPS ≥1% was lower in LBCs than in CBs (33 of 60 [55.0%] vs. 35 of 60 [58.3%]; p = .687). Even with the TPS ≥50% threshold, PD-L1 expression was lower in LBCs (10 of 60 [16.7%] vs. 15 of 60 [25%]; p = .125). Epidermal growth factor receptor (EGFR) exon 20 mutation, tumor cell proportion, and pleural fluid neutrophil-to-lymphocyte ratio were related to PD-L1 expression on CBs (p = .013, p = 0.022, and p = .011), respectively. Patients with subsequent immune checkpoint inhibitor therapy remained a better prognostic in subgroups of PD-L1 positive expression on LBC slides (TPS ≥1%, p = .041). CONCLUSIONS: LBC specimens had comparable performance to CBs in PD-L1 assessment and predicting treatment response to PD-L1-defined therapy.

Molecular interactions of antibodies with PD-1/PD-L1 proteins.

Aim: To compare the protein-protein interactions of antibodies targeting PD-1 and its ligand (PD-L1) with their targets in an attempt to explain the antibodies' binding affinity. Materials & methods: The structural features of complexes between pembrolizumab, nivolumab, durvalumab, atezolizumab, avelumab and PD-1/PD-L1 are described, with the use of software and based on crystallographic data. Results: Pembrolizumab has more structural features, including the number and type of the bonds and total binding surface area, which could rationalize its different clinical behavior compared with nivolumab. Similarly, protein-protein interactions with PD-L1 differ among durvalumab, atezolizumab and avelumab. Conclusion: Differential protein-protein interactions between antibodies and PD-1/PD-L1 may indicate differential clinical activity; however, further research is needed to provide evidence.

This study looked at different immunotherapy drugs used to treat cancer. These drugs bind to two different proteins, called PD-1 and PD-L1, that are part of our immune system. These proteins usually act as brakes in our immune system. The drugs block the brakes, which boosts the immune system and improves the immune defense against cancer. Using computer images, the authors compared how each drug binds to PD-1/PD-L1. The results showed that these drugs bind to PD-1 and PD-L1 with different chemical bonds. These bonds can be smaller or larger depending on the drug. The drugs' different chemical bonds with PD-1/PD-L1 might show that they do not act exactly the same when they are given to patients. However, further studies are needed for more information.

Identification of the functional PD-L1 interface region responsible for PD-1 binding and initiation of PD-1 signaling.

The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.

Computational design of PD-L1 small molecule inhibitors for cancer therapy.

Drug repurposing opens new avenues in cancer therapy. Drug repurposing, or finding new uses for existing drugs, can substantially reduce drug discovery time and costs. Cheminformatics, genetics, and systems biology advances enable repositioning drugs. Clinical usage of PD-1/PD-L1 blocking has been approved because of its efficacy in improving prognosis in select groups. The PD-1/PD-L1 axis was considered to represent a mechanism for tumour evasion of host tumour antigen-specific T-cell immunity in early preclinical research. The expression of PD-L1 in cancer cells causes T lymphocytes to become exhausted by transmitting a co-inhibitory signal. A better understanding of how PD-L1 is regulated in cancer cells could lead to new therapeutic options. In this view, the study was aimed to repurpose the existing FDA-approved drugs as a potential PD-L1 inhibitor through e-Pharmacophore modelling, molecular docking and dynamic simulation. e-Pharmacophore screening retrieved 324 FDA-approved medications with the fitness score ≥ 1. The top 10-docked FDA candidates were compared with IN-35 (Clinical trial candidate) for its interaction pattern with critical amino acid residues. Mirabegron and Indacaterol exhibited a greater affinity for PD-L1 with docking scores of - 9.213 kcal mol-1 and - 8.023 kcal mol-1, respectively. Mirabegron retain interactions at all three major hotspots in the PD-L1 dimer interface similar to IN-35. MM-GBSA analyses indicated that Mirabegron uses less energy to create a more stable complex and retains all of the inhibitor's positive interactions found in clinical trial ligand IN-35. Molecular dynamics simulation analysis of the Mirabegron complex showed a similar pattern of deviation in correlation with IN-35, and it retains the interaction with the active key amino acids throughout the simulation time. Our present study has shown Mirabegron as a powerful inhibitor of PD-L1 expression in cancer cells using a drug-repurposing screen.

Computational discovery of small drug-like compounds as potential inhibitors of PD-1/PD-L1 interactions.

The programmed cell death ligand protein 1 (PD-L1) is a strong immunosuppressive molecule that inactivates tumor-specific T cells by binding to the programmed cell death- 1 protein (PD-1). Cancer immunotherapy based on the monoclonal antibodies targeting the PD-1/PD-L1 pathway has demonstrated therapeutic responses without precedent over a wide range of cancers. However, the antibody-based immunotherapies have several limitations such as high production cost or the induction of severe immune-related adverse effects. Small-molecule inhibitors of the PD-1/PD-L1 pathway are a promising alternative or complementary therapeutic to antibodies. Currently, the field of developing anti-PD-1/PD-L1 small-molecule inhibitors is intensively explored. In the present study a pharmacophore model was generated based on previously developed compounds and their atomistic structures with the PD-L1 dimer. Structure-based affinity-based virtual screening of small-molecule inhibitors of the PD-1/PD-L1 pathway according to the pharmacophore model followed by a screening in terms of drug-likeness resulted in ten hit compounds of high affinity towards the PD-L1 dimer and the satisfaction to all of the drug-likeness rules. Molecular dynamics (MD) simulations showed that nine of ten compounds formed stable complexes with the PD-L1 dimer as evidenced by the analysis of MD trajectories. Molecular mechanics Poisson- Boltzmann surface area (MM-PBSA) calculation revealed very low binding energies (<-46 kcal/mol) for the interactions of these ligands with the PD-L1 dimer, suggesting that identified compounds may serve as good scaffolds for the design of novel agents of antitumor immunotherapy able to target the PD-1/PD-L1 interactionCommunicated by Ramaswamy H. Sarma.

Exploiting the Innate Plasticity of the Programmed Cell Death-1 (PD1) Receptor to Design Pembrolizumab H3 Loop Mimics.

Checkpoint blockade of the immunoreceptor programmed cell death-1 (PD1) with its ligand-1 (PDL1) by monoclonal antibodies such as pembrolizumab provided compelling clinical results in various cancer types, yet the molecular mechanism by which this drug blocks the PD1/PDL1 interface remains unclear. To address this question, we examined the conformational motion of PD1 associated with the binding of pembrolizumab. Our results revealed that the innate plasticity of both C'D and FG loops is crucial to form a deep binding groove (371 Å3 ) across several distant epitopes of PD1. This analysis ultimately provided a rational-design to create pembrolizumab H3 loop mimics [RDYRFDMGFD] into ß-hairpin scaffolds. As a result, a 20-residue long ß-hairpin peptide 1 e was identified as a first-in-class potent PD1-inhibitor (EC50 of 0.29 µM; Ki of 41 nM).

Discovery of 4-phenylindolines containing a (5-cyanopyridin-3-yl)methoxy moiety as potent inhibitors of the PD-1/PD-L1 interaction.

With the great success of anti-programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) monoclonal antibodies in clinical applications, blocking the PD-1/PD-L1 pathway has become the most compelling strategy in the field of tumor immunotherapy. In this study, a novel series of 4-phenylindolines containing a (5-cyanopyridin-3-yl)methoxy moiety were developed, and their structure-activity relationships were preliminarily discussed. Among them, compounds M17 and M23 exhibited the most potent ability to disrupt the PD-1/PD-L1 interaction, demonstrating IC50 values of 60.1 nM and 53.2 nM, respectively. The binding mode of M23 was further explored by molecular docking analysis with dimeric PD-L1. Therefore, M17 and M23 are promising lead compounds for developing potent inhibitors of the PD-1/PD-L1 axis.

Unraveling a Conserved Conformation of the FG Loop upon the Binding of Natural Ligands to the Human and Murine PD1.

The activation of T cells is normally accompanied by inhibitory mechanisms within which the PD1 receptor stands out. PD1 drives T cells to an unresponsive state called exhaustion, characterized by a markedly decreased capacity to exert effector functions upon binding the ligands PDL1 and PDL2. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling pathway is activated upon the ligands' binding remains an open question. In this work, we used molecular dynamics simulations to assess the differences of the PD1 motion in the free state and in complex with the ligands. We found that, in both human and murine systems, the binding of PDL1 and PDL2 stabilizes the conformation of the FG loop similarly. This result, combined with the conservation of the FG loop residues across species, suggests that the conformation of the FG loop is somehow related to the signaling process. We also found a high similarity between the PD1-PDL1 structures with the variable region of an antibody structure, where the FG loop occupies a similar position to the CDR3 light chain.

Insights into small molecule inhibitor bindings to PD-L1 with residue-specific binding free energy calculation.

Targeting the immunological checkpoint PD-1/PD-L1 with antibodies has shown opportunities to improve cancer treatment in recent years. However, antibody therapy is a double-edged sword with high cost, low patient tolerance, lack of oral bioavailability, and a reaction to most solid tumors that prevents the adoption of antibodies. Advancement of small-molecule PD-1/PD-L1 inhibitors that could overwhelm these drawbacks is sluggish because of the poor pharmacodynamic properties and shallow pocket of the PD-1/PD-L1 binding interface. Recently, a number of compounds have been discovered to bind the PD-L1/PD-L1 dimer interface, providing an excellent alternative to inhibit the interaction between PD-1/PD-L1 and small molecules. Quantitative characterization of PD-L1 interactions with these inhibitors will advance the design of novel and efficient inhibitors in the future. Here, the binding free energies of 35 PD-L1 dimer inhibitors have been calculated using the alanine-scanning-interaction-entropy (AS-IE) method. Hotspot residues on PD-L1 and potential modification groups on the inhibitors were identified. The experimental results for the AS-IE method were better correlated than the classical MM/GBSA method. These results may set the stage for the design the more powerful PD-L1 inhibitors.Communicated by Ramaswamy H. Sarma.

Design, synthesis and biological evaluation of isoxazole-containing biphenyl derivatives as small-molecule inhibitors targeting the programmed cell death-1/ programmed cell death-ligand 1 immune checkpoint.

Monoclonal antibodies targeting the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) immune checkpoint have achieved enormous success in cancer immunotherapy. But the antibody-based immunotherapies carry a number of unavoidable deficiencies such as poor pharmacokinetic properties and immunogenicity. Small-molecule PD-1/PD-L1 inhibitors offer the superiority of complementarity with monoclonal antibodies and represent an appealing alternative. A novel series of isoxazole-containing biphenyl compounds were designed, synthesized and evaluated as PD-1/PD-L1 inhibitors in this paper. The structure-activity relationship of the novel synthesized compounds indicated that the ring-closure strategy of introducing isoxazole could be employed and the 3-cyanobenzyl group was significant for the inhibitory activity against the PD-1/PD-L1 protein-protein interactions. Molecular docking studies were performed to help understand the binding mode of the small-molecule inhibitor with the PD-L1 dimer. In particular, compound II-12 was a promising anti-PD-1/PD-L1 inhibitor with the IC50 value of 23.0 nM, providing valuable information for future drug development.

A mating mechanism to generate diversity for the Darwinian selection of DNA-encoded synthetic molecules.

DNA-encoded library technologies enable the screening of synthetic molecules but have thus far not tapped into the power of Darwinian selection with iterative cycles of selection, amplification and diversification. Here we report a simple strategy to rapidly assemble libraries of conformationally constrained peptides that are paired in a combinatorial fashion (suprabodies). We demonstrate that the pairing can be shuffled after each amplification cycle in a process similar to DNA shuffling or mating to regenerate diversity. Using simulations, we show the benefits of this recombination in yielding a more accurate correlation of selection fitness with affinity after multiple rounds of selection, particularly if the starting library is heterogeneous in the concentration of its members. The method was validated with selections against streptavidin and applied to the discovery of PD-L1 binders. We further demonstrate that the binding of self-assembled suprabodies can be recapitulated by smaller (∼7 kDa) synthetic products that maintain the conformational constraint of the peptides.

A simple, rapid and low-cost qPCR assay for evaluating the severity of exosomal PD-L1-mediated T cell exhaustion in blood samples.

A novel dual-aptamer activated proximity-induced qPCR assay was developed for the quantitative analysis of exosomal PD-L1 on T cell-exosome complexes in blood samples.

Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

USP5 facilitates non-small cell lung cancer progression through stabilization of PD-L1.

PD-L1(CD274) is a well-known immunosuppressive molecule, which confers immunoescape features to cancer cells and has become one of the major targets in cancer immunotherapies. Understanding the regulatory mechanisms that control PD-L1 protein expression is important for guiding immune checkpoint blockade therapy. Here, we showed that ubiquitin specific peptidase 5 (USP5) was a novel PD-L1 deubiquitinase in non-small cell lung cancer (NSCLC) cells. USP5 directly interacted with PD-L1 and deubiquitinated PD-L1, therefore enhances PD-L1 protein stability. Meanwhile, USP5 protein levels were highly elevated and positively correlated to PD-L1 levels in NSCLC tissues, and were closely correlated with poor prognosis of these patients. In addition, knockdown of USP5 retarded tumor growth in the Lewis lung carcinoma mouse model. Thus, we identified that USP5 was a new regulator of PD-L1 and targeting USP5 is a promising strategy for cancer therapy.

Molecular Mechanism of Food-Derived Polyphenols on PD-L1 Dimerization: A Molecular Dynamics Simulation Study.

In cancer immunotherapy, an emerging approach is to block the interactions of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) using small-molecule inhibitors. The food-derived polyphenols curcumin (CC), resveratrol (RSV) and epigallocatechin gallate (EGCG) have anticancer immunologic functions, which, recently, have been proposed to act via the downregulation of PD-L1 expression. However, it remains unclear whether they can directly target PD-L1 dimerization and, thus, interrupt the PD-1/PD-L1 pathway. To elucidate the molecular mechanism of such compounds on PD-L1 dimerization, molecular docking and nanosecond molecular dynamics simulations were performed. Binding free energy calculations show that the affinities of CC, RSV and EGCG to the PD-L1 dimer follow a trend of CC > RSV > EGCG. Hence, CC is the most effective inhibitor of the PD-1/PD-L1 pathway. Analysis on contact numbers, nonbonded interactions and residue energy decomposition indicate that such compounds mainly interact with the C-, F- and G-sheet fragments of the PD-L1 dimer, which are involved in interactions with PD-1. More importantly, nonpolar interactions between these compounds and the key residues Ile54, Tyr56, Met115, Ala121 and Tyr123 play a dominant role in binding. Free energy landscape and secondary structure analyses further demonstrate that such compounds can stably interact with the binding domain of the PD-L1 dimer. The results provide evidence that CC, RSV and EGCG can inhibit PD-1/PD-L1 interactions by directly targeting PD-L1 dimerization. This provides a novel approach to discovering food-derived small-molecule inhibitors of the PD-1/PD-L1 pathway with potential applications in cancer immunotherapy.

Targeting cryptic-orthosteric site of PD-L1 for inhibitor identification using structure-guided approach.

Approved mAbs that block the protein-protein interaction (PPI) interface of the PD-1/PD-L1 immune checkpoint axis have led to significant improvements in cancer treatment. Despite having drawbacks of mAbs only few a compounds are reported till date against this axis. Inhibiting PPIs using small molecules has emerged as a significant therapeutic opportunity, demanding for the identification of drug-like molecules at an accelerated pace under the hit-to-lead campaigns. Due to the PD-L1's cross-talk with PD-1/CD80 and its overexpression on cancer cells, as well as the availability of its crystal structures with small molecules, it is an enticing therapeutic target for structure-assisted small molecule design. Furthermore, the selection of chemical databases enriched with focused designing for PPI interfaces is crucial. Therefore, in this study we have utilized the Asinex signature library for structure-assisted virtual screening to find the potential PD-L1 inhibitors by targeting the cryptic PD-L1 interface, followed by induced fit docking for pose refinements in the pocket. The obtained hits were then subjected to interaction fingerprinting and ligand-based drug-likeness investigations in order to evaluate and analyze their drug-like qualities (ADME). Twelve compounds qualified for molecular dynamics simulations, followed by thermodynamic calculations for evaluation of their stability and energetics inside the pocket. Two novel compounds with different chemical moieties have been identified that are consistent throughout the simulation, mimicking the interactions and binding energies with BMS-1166. These compounds appear as potential therapeutic candidates to be explored experimentally, thereby paving the way for the development of novel leads as immunomodulators.

Allicin May Promote Reversal of T-Cell Dysfunction in Periodontitis via the PD-1 Pathway.

We evaluated the role of allicin in periodontitis using an in silico and in vitro design. An in silico docking analysis was performed to assess the plausible interactions between allicin and PD-L1. The cytokine profile of gingival crevicular fluid (GCF) samples obtained from periodontitis patients was estimated by cytometric bead array. CD3+ lymphocytes isolated from the peripheral blood were sorted and characterized using immunomagnetic techniques. Cultured and expanded lymphocytes were treated with the GCF samples to induce T-cell exhaustion. Optimum concentrations of allicin were added to exhausted lymphocytes to compare the expression of TIM-3 and LAG-3 gene expression at baseline and post-treatment. Allicin was found to bind to the PD-L1 molecule as revealed by the in-silico experiment, which is possibly an inhibitory interaction although not proven. GCF from periodontitis patients had significantly higher concentrations of TNF-α, CCL2, IL-6, IFN-γ, and CXCL8 than controls. GCF treatment of CD3+ lymphocytes from the periodontitis patients significantly increased expression of T-cell exhaustion markers TIM-3 and LAG-3. Allicin administration with GCF treatment resulted in significant lowering of the expression of exhaustion markers. Allicin may exert an immunostimulatory role and reverse immune-destructive mechanisms such as T-cell exhaustion.