Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. It has commonly been associated with exposure to tobacco-derived carcinogens and alcohol consumption. Pembrolizumab has shown to be effective in the treatment of many types of cancers such as melanoma, non-small cell lung cancer, due to its antiproliferative, immunoregulatory properties. The aim of this study was to investigate the effects of naked Pembrolizumab and Pembrolizumab liposome on the level of DNA damage, gene, and protein expressions in peripheral lymphocytes from HNC patients and compared to the healthy individuals by using the Comet and micronucleus assays. Western blotting and real-time polymerase chain reaction were performed to assess the potential of improving the repair mechanisms after treatment with naked Pembrolizumab and Pembrolizumab liposome. According to the results, Comet assay and micronucleus assay showed a significantly decreased DNA damage in the lymphocytes from HNC patients after being treated with naked Pembrolizumab and pembrolizumab liposome. Furthermore, the results have shown that naked Pembrolizumab and pembrolizumab liposomes (10 μg/ml) greatly decreased the oxidative stress produced by H2O2. Both forms of pembrolizumab have also demonstrated improving the repair mechanisms in lymphocytes from HNC patients by modulating the expression of P53, P21, and Bcl-2 at mRNA and protein levels. This study suggested that Pembrolizumab naked and liposome could have an antioxidant role alongside other actions in the treatment of HNSCC. However, further studies on Cancer cell lines and in vivo observation are required to validate the anticancer potential of pembrolizumab naked with liposome in HNC. / Ministry of Higher Education and Scientific Research, Libya

About 12 to 17 percent of all breast cancers are triple-negative breast cancer (TNBC). In TNBC, estrogen, progesterone, and human epidermal growth factor receptor two are not expressed, or copies of the HER2 gene are decreased (or both). This makes TNBC hard to treat in comparison with other kinds of breast cancers. New studies have made some interesting observations on how some monoclonal antibodies can help to treat TNBC. One of the monoclonal antibodies that can be useful for treating TNBC is pembrolizumab. Pembrolizumab inhibits programmed death ligand 1 (PD-1), which is located on the surface of T cells from connecting to immune checkpoint proteins such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) located on the surface of the cancer cell. Breaking this connection enables continued activation of T cells and attack of the cancer cells. This thesis aimed to evaluate if pembrolizumab was safe and effective as monotherapy or as a combination therapy with chemotherapy for patients with different stages of TNBC. This study was based on scientific articles identified from the database PubMed. Five randomized controlled trial studies were selected for further study in this project. Two publications were chosen from keynote-355, which studied the effect and safety of Pembrolizumab in combination with chemotherapy and compared it to chemotherapy monotherapy in patients with metastatic TNBC. The first study included patients from different countries as it was a multi-center study, and the second one focused only on patients who enrolled in Japan. One study was chosen from keynote-119 studies, which compared health-related quality of life for patients who were treated with pembrolizumab monotherapy or with monotherapy of chemotherapy. The last two articles which were chosen were about keynote-522. The first article about keynote-522 compared was a multicenter study enrolled in 21 countries. In this study, patients had early-stage TNBC and received neoadjuvant placebo chemotherapy or pembrolizumab chemotherapy. After the breast operation, either adjuvant pembrolizumab or placebo was received. The other study looked at Asian patients who enrolled in keynote-522. Results showed that monotherapy with pembrolizumab did not make a massive difference in overall survival compared to chemotherapy. Still, it led to better health- related quality of life for patients (Combined Positive Score (CPS) ≥ 10) treated with pembrolizumab. Results from keynote-355 showed that combination therapy with pembrolizumab and chemotherapy led to better and longer progression-free survival and overall survival in patients with CPS ≥ 10 treated with pembrolizumab. The analysis of Japanese patients showed even better progression-free survival and overall survival results than the global population. The study from keynote-522 showed that neoadjuvant pembrolizumab and chemotherapy followed by adjuvant pembrolizumab had a better effect than only neoadjuvant chemotherapy. The second keynote-522 study showed the same results as the global study and better results at event-free survival for the Asian population than the overall population. After reviewing the articles, it was found that pembrolizumab proves to be a safe and effective treatment for TNBC. To enhance understanding of the drug's effects, measures such as extending follow-up periods, conducting further studies to assess its effectiveness, and exploring new research methodologies are proposed.

PD-1 (Programmed Cell death protein -1) est un récepteur co-inhibiteur exprimé à la surface de lymphocytes T (LT) activés. Ce récepteur joue un rôle important dans le maintien de la tolérance périphérique tout en protégeant contre les maladies auto-immunes et inflammatoires. Cependant, une expression élevée et permanente de PD-1 et ses ligands PD-L1 et PD-L2 (PD-Ls) perturbe la réponse immunitaire contre les pathogènes et les cellules tumorales. Les inhibiteurs de points de contrôle immunitaires (ICI) ciblant l'axe PD-1/PD-Ls représentent aujourd’hui une avancé majeure dans le traitement de différents types de cancer, tant sur le plan de l’efficacité que de la tolérance. Le nivolumab (nivo) et le pembrolizumab (pembro) sont deux anticorps monoclonaux (AcM) anti-PD-1 qui bloquent l’interaction de PD-1 avec ses ligands. Ces AcM ont montré des résultats prometteurs dans le traitement de multiples types de cancers comme le mélanome, le cancer bronchique non à petites cellules, le carcinome à cellules rénales, etc. Malgré l’importance thérapeutique de nivo et de pembro dans le cancer, aucune étude n’a défini le devenir de PD-1 après la liaison à ces deux AcM. L’objectif de cette étude a été donc d’évaluer l’endocytose de PD-1 suite au liaison à des AcM anti-PD-1 (clone J110, nivo, pembro) et de déterminer s’il y a différence entre nivo et pembro vis à vis leur capacités à induire l'internalisation de PD-1. L’étude de l’endocytose de PD-1 a été réalisé sur des LT humains obtenus à partir du sang périphérique de donneurs sains et activés avec des Ac anti-CD3/anti-CD28 ou concanavaline A afin d’exprimer le récepteur PD-1. L’analyse des données par cytométrie en flux a montré que l’engagement de PD-1 avec l’Ac anti-PD-1 (clone J110) induit son endocytose dans les LT humains et que 50% de la totalité de PD-1 de surface est internalisé dans les premiers 30 minutes suivant l’incubation de cellules à 37°C, suivi d’un taux d’endocytose plus lent (56% en 60min). Notre étude a montré également que la liaison de nivo et de pembro au PD-1 induit son endocytose et que la plupart de récepteur est internalisée dans les 30 min suivant l’incubation de cellules à 37°C. Toutefois, 32 à 50% des récepteurs sont résistants à l’endocytose. L’analyse comparative de nivo et de pembro a révélé une différence statistiquement significative (p=0.03) entre le taux d’internalisation du complexe PD-1/nivo et celui du PD-1/pembro (46% versus 25% en 30min, respectivement). Même à des concentrations élevées de pembro, la liaison de nivo induit une meilleure internalisation de PD-1, ce qui suggère que nivo pourrait être plus efficace. Bien que les ICI comme nivo et pembro sont connus de bloquer l’interaction de PD-1 avec ses ligands, PD-L1 et PD-L2, notre étude a montré pour la première fois que ce deux AcM induisent aussi l’endocytose du récepteur PD-1 dans les LT humains avec des taux différents, et que 32% à 50% de récepteurs PD-1 sont résistants à l’endocytose. Ces résultats pourraient être exploités pour améliorer l’internalisation de PD-1 dans les LT humains et par la suite augmenter les potentiels thérapeutiques de nivolumab et de pembrolizumab dans le traitement du cancer. Mots clés : Récepteur PD-1, Ligands de PD-1, Lymphocytes T, Inhibiteurs de point de contrôle immunitaires, Anticorps anti-PD-1, Nivolumab, Pembrolizumab, Endocytose, Cancer / PD-1 (Programmed Cell death protein -1) is a co-inhibitory receptor expressed on the surface of activated T cells. It plays an important role in maintaining peripheral tolerance and protecting against autoimmune and inflammatory diseases. However, permanent expression of PD-1 and its ligands PD-L1/ PD-L2 (PD-Ls) disrupts the immune response against pathogens and tumor cells. Immune checkpoint blockade (ICB) targeting the PD-1/PD-Ls axis has revolutionized the treatment of many cancers. Nivolumab (nivo) and pembrolizumab (pembro) are two anti-PD-1 monoclonal antibodies (mAb) that block the interaction between PD-1 and its ligands. They have shown promising results in the treatment of multiple types of cancers such as melanoma, non-small cell lung cancer, renal cell carcinoma, etc. Surprisingly, despite the success of anti-PD-1 in cancer immunotherapy, no-one has defined the destiny of surface PD-1 following antibody binding. Therefore, the objective of my master thesis was to define the fate of surface PD-1 following antibody binding and whether different anti-PD-1 Abs in the clinic differ in their ability to induce PD-1 endocytosis. The study of PD-1 endocytosis was performed on human T lymphocytes obtained from peripheral blood of healthy donors and activated with anti-CD3/anti-CD28 Ab or concanavalin A to express PD-1 receptor. Data analysis by flow cytometry showed that following anti-PD-1 Ab binding, 50% of PD-1 becomes endocytosed by 30min. In addition, we found that the PD-1 receptor is internalised upon its engagement with nivo and pembro and that most of the receptor is endocytosed within 30 min. However, 32 to 50% of the receptors are resistant to endocytosis. The comparative analysis of nivo and pembro has revealed a statistically significant difference (p=0.03) between the internalisation rate of the PD-1/nivo complex versus PD-1/pembro (46% versus 25% by 30min, respectively). Even at high concentrations of pembro, nivo induces better internalization of PD-1, suggesting that nivo could be more effective than pembro. Our study showed for the first time that ICB involves not only in the blockade of PD-1/PD-Ls interaction, but also in the endocytosis of PD-1 receptors from the surface of human T-cells, which differs between nivolumab and pembrolizumab. These results could be exploited to increase the therapeutic potential of nivolumab and pembrolizumab in cancer treatment. Keywords: PD-1 receptor, PD-1 ligands, T lymphocytes, Immune checkpoint blockade, Anti-PD1 antibodies, Nivolumab, Pembrolizumab, Endocytosis, Cancer

L’avènement des inhibiteurs du point de contrôle immunitaire (ICIs) ciblant l’axe PD-1/PD-L1 a révolutionné le traitement des patients avec un carcinome pulmonaire non à petites cellules (CPNPC). Ce mémoire consolide les conclusions de trois études distinctes visant à analyser et à améliorer l'efficacité des ICIs en monothérapie chez le CPNPC. La première section explore les bénéfices associés au durvalumab suivant une chimioradiothérapie chez les patients présentant un CPNPC localement avancé, confirmant le bénéfice de survie associé aux ICI et élargissant les perspectives émises depuis l'étude PACIFIC, y compris au niveau de la valeur prédictive du PD-L1. Dans l’optique de caractériser de nouveaux biomarqueurs d’efficacité, la deuxième section souligne le rôle crucial du microbiome intestinal dans la modulation de la réponse aux ICIs, spécifiquement au niveau de la dysbiose intestinale liée aux antibiotiques. La méta-analyse proposée confirme l’impact délétère des antibiotiques sur la survie des patients traités avec un ICI, tout particulièrement lorsque l’antibiothérapie précède l’inhibition du PD-1/PD-L1. Dans le domaine des stratégies immunomodulatrices émergentes, la troisième section explore l’impact de la cryoablation chez les patients présentant un CPNPC avec PD-L1≥50% et traités avec le pembrolizumab. Sans relever un signal d’efficacité supérieure, cette étude de phase I/II confirme la faisabilité et l’innocuité de la cryoablation, une technique permettant la libération d’antigènes tumoraux en circulation sans dénaturation thermique. Ultimement, ce mémoire propose un survol des bénéfices de survie, biomarqueurs prédictifs et stratégies immunomodulatrices liés à l’utilisation des ICIs chez les patients avec un CPNPC avec l’espoir d’optimiser les paradigmes thérapeutiques existants. / The advent of immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis has revolutionized the therapeutic landscape for patients diagnosed with non-small cell lung cancer (NSCLC). This thesis consolidates the findings of three distinct studies aiming to analyze and enhance the efficacy of ICI monotherapy in NSCLC. The first section delves into the real-world use of durvalumab following chemoradiotherapy in stage III NSCLC, confirming the survival benefit associated with ICI administration in this context and broadening the insights derived from the PACIFIC study, particularly regarding the predictive value of PD-L1. With the aim of characterizing new biomarkers of efficacy, the second section sheds light on the crucial role of the gut microbiome in modulating responses to ICIs, particularly intestinal dysbiosis related to antibiotics. The meta-analysis confirms the detrimental impact of antibiotics on the overall survival of patients with advanced cancer treated with ICI monotherapy, especially when antibiotic therapy precedes PD-1/PD-L1 inhibition. In the realm of emerging immunomodulatory strategies, the third section explores the impact of cryoablation in patients with NSCLC and PD-L1≥50% treated with pembrolizumab. Although the procedure did not translate into a signal of superior efficacy, the proposed phase I/II study confirms the feasibility and safety of cryoablation, a technique allowing the release of circulating tumor antigens without heat-related denaturation. Ultimately, this thesis presents a contemporary overview of survival benefits, predictive biomarkers, and immunomodulatory strategies associated with the use of ICIs in monotherapy in patients with NSCLC, with the hope of optimizing existing therapeutic paradigms.