Dissertations / Theses on the topic 'Immune checkpoint inhibitors (ICIs)'

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Background: Clinical trial completion is critical for new cancer therapies. Premature trial termination or withdrawal is common and impairs progress. We assessed factors of early terminated/withdrawn oncology trials focusing on trials with immune checkpoint inhibitors (ICI), hypothesizing that the latter may be associated with lower rates of premature discontinuation. Materials and Methods: We reviewed all adult, intervention, oncology trials registered in ClinicalTrials.gov (November 16, 2011, to April 16, 2015) to identify all terminated/withdrawn trials and reasons for termination. Logistics regression model was used to identify factors associated with early termination/withdrawal. Discontinuation rate was compared in trials with and without ICI. Results: We identified 12,875 trials (35% industry funded, 12% federal funded), of which 8.5% were prematurely terminated (5%) or withdrawn (3.5%); the main reasons were poor accrual (33%) and logistical (24%). ICI trials (n = 350) had a nonsignificant lower rate of termination or withdrawal compared with all other oncology trials (5.4% vs. 8.5%; p =.9) and were less likely to discontinue due to poor accrual (nonsignificant difference: 21% vs. 33%; p =.4). ICI trials were also less likely to discontinue compared with all other oncology drug trials (e.g., chemotherapy, targeted inhibitors, antiangiogenesis, biologics; 5.4% vs. 7.9%, respectively, nonsignificant difference). The 4-year cumulative incidence of failing to complete for reasons unrelated to toxicity or efficacy was 18% (95% confidence interval 16%–20%). There was no association between annual incidence across different tumor types or accrual goal and rate of trial termination. Conclusion: Poor accrual represents the main cause of early cancer trial termination. Premature termination/withdrawal rate was not significantly lower in ICI compared with other trials. Clinical trial completion remains a high priority and can be influenced by provider and patient factors. Implications for Practice: Clinical trial completion is critical for new cancer therapies. Premature trial termination or withdrawal is common and impairs progress. This study assessed factors of early terminated/withdrawn oncology trials, focusing on trials with immune checkpoint inhibitors (ICI), and found that poor accrual represents the main cause of early cancer trial termination. Premature termination/withdrawal rate was not significantly lower in immune checkpoint inhibitor trials compared to other trials. The discussion herein is focused on measures taken by the National Cancer Institute and other institutions to improve clinical trial accrual and prevent premature clinical trial discontinuation.
Revisión por pares

Precision medicine has allowed for the development of monoclonal antibodies that unmask the anti-tumor immune response. These agents have provided some patients durable clinical benefit. However, PD-1 and PD-L1 inhibitor therapies are effective in a small group (10-20%) of non-small cell lung cancer (NSCLC) patients when used as single-agent therapy. The approved companion diagnostic is expression of the immune cell surface molecule, programmed death ligand 1 (PD-L1), on tumors measured by immunohistochemistry (IHC). Studies in tumor biology and immune surveillance dictate that PD-1 inhibitor efficacy should depend on the level of PD-L1 expression; however, the literature has not followed with convincing evidence. The limitations of this test include timing of tissue acquisition, tumor heterogeneity, and timing of therapy relative to the expression of PD-L1. In addition, the requirement of analyzing tumor tissue biopsy samples from a patient is cumbersome. Thus, a peripheral blood biomarker that predicts efficacy of PD-1/PD-L1 inhibition would be optimal for precise and cost-effective treatment. A history of chronic inflammatory diseases may be advantageous for a cancer patient who is treated with PD-1/PD-L1 inhibitors and may allow them to then mobilize a swift immune response to tumor cells. Specific biological components of this persistent inflammation may predict PD-1 inhibitor response. We have taken a novel approach to leverage national healthcare claims data that couples patient history with response to therapy. We have identified potential peripheral blood biomarkers of response to PD-1/PD-L1 inhibitors using a combination of healthcare outcomes and molecular markers that correlate with therapeutic efficacy.

Les découvertes scientifiques liées à la radiothérapie sont en progression continue et près de 60 % des patients diagnostiqués d'un cancer sont traités par radiothérapie. Cependant, ce traitement fait face à de nombreuses limitations dues à la radiorésistance et aux effets secondaires infligés sur les tissus sains. Pour surmonter ces problèmes, un intérêt particulier a été suscité sur le rôle que pourrait jouer la nanomédecine dans l'amélioration de la réponse anti-tumorale médiée par le système immunitaire. Dans ce contexte, nous avons décidé d'évaluer la capacité de la combinaison de nanoparticules de Gadolinium (AGuIX) avec les rayonnements ionisants (RI) à stimuler une réponse immunologique anti-tumorale et à augmenter l'efficacité de la radiothérapie associée à des inhibiteurs de points de contrôle immunitaires. AGuIX sont de très petites nanoparticules composées d'une matrice de polysiloxane et de chélates de gadolinium. Grâce aux propriétés magnétiques du Gadolinium, ces nanoparticules jouent le rôle d'agent de contraste, en plus de ses propriétés radiosensibilisantes, permettant ainsi un meilleur ciblage et un index thérapeutique renforcé.Nos travaux révèlent la capacité de AGuIX + RI à induire une instabilité génomique et à stimuler les voies de signalisation cellulaire immunomodulatrices. En parallèle, nous démontrons le potentiel anti-tumoral de la combinaison d'AGuIX + RI en évaluant la croissance tumorale et la survie globale à l'aide d'un modèle préclinique de souris immunocompétentes porteuses de tumeurs. Nos résultats suggèrent un effet synergique de la combinaison d'AGuIX + RI sur la croissance tumorale en stimulant le système immunitaire. Enfin, nous révélons la capacité de la combinaison d'AGuIX + RI à surmonter la résistance aux inhibiteurs de points de contrôle immunitaires. En conclusion, nos travaux montrent que la combinaison d'AGuIX + RI stimule des voies de signalisation immunomodulatrices, induit une forte réponse anti-tumorale synergique et permet de surmonter la résistance aux immunothérapies
Scientific discoveries linked to radiotherapy are in continuous progress and almost 60% of cancer diagnosed patients are treated with radiotherapy. However, radiation therapy still faces many limitations due to radioresistance and the side effects inflected on healthy tissues. To overcome these factors, a particular interest has been aroused on the role that nanomedicine could play in the improvement of immune-mediated anti-tumor response. In this context, we decided to assess the ability of the combination of Gadolinium nanoparticles (AGuIX) with ionizing radiation (IR) to stimulate an anti-tumor immunological response and to increase the effectiveness of radiotherapy combined to immune checkpoint blockers.AGuIX are very small nanoparticles composed of a polysiloxane matrix and gadolinium chelates. Thanks to the magnetic properties of gadolinium, these nanoparticles play the role of a contrast agent, in addition to its radiosensitizing properties, thus allowing a better targeting and an enhanced therapeutic index.Our work reveals the ability of the AGuIX+IR combination to induce genomic instability and to stimulate immunomodulatory cell signaling pathways in treated human and murine cancer cells. In parallel, we demonstrate the anti-tumor activity of this combination by assessing tumor growth and overall survival using a preclinical model of immunocompetent mice bearing tumors. Our results strongly demonstrate a synergistic effect of AGuIX + IR combination on tumor growth and overall survival by stimulating the immune system. Finally, we reveal the capacity of AGuIX + IR combination to overcome immune checkpoint blockers resistance.In conclusion, our work shows that the combination of AGuIX+IR stimulates immunomodulatory signaling pathways, induces a strong synergistic anti-tumor response and helps overcoming resistance to immunotherapies

Histone deacetylases (HDACs) are key mediators of gene expression and, thus, major regulators of cell function. As such, HDACs play a role in orchestrating tumor biology, and the use of small inhibitors targeting theses proteins is attractive for the field of cancer therapy. Indeed, several HDAC inhibitors have received FDA-approval for the treatment of malignancies, while a myriad of these compounds continue to be evaluated in clinical trials. Besides their direct impact on tumor growth, HDAC inhibitors have been shown to increase immunogenicity of cancer cells, facilitating generation of a productive immune response against tumors. Immunotherapeutic approaches take advantage of the intrinsic ability of the immune system to manifest an anti-tumor response. Mechanisms of immune escape are often developed by cancer cells, neutralizing activity of the immune system. For example, upregulation of the PD1 ligands PDL1 and PDL2 by tumor cells negatively regulates the anti-tumor functions of PD1-expressing infiltrating T-cells. Importantly, strategies targeting this inhibitory axis have shown outstanding clinical benefit for the treatment of solid and hematological malignancies. The mechanisms by which HDAC inhibitors modulate tumor and immune cells biology were explored herein. Initially, treatment of melanoma cells with pan- and class I-selective HDAC inhibitors resulted in upregulation of PDL1 and PDL2 molecules. These effects were observed in mouse and human cell lines, as well as in tumor cells resected from metastatic melanoma patients. This upregulation was robust and sustained, lasting at least 96 hours in vitro, and validated in vivo using a B16F10 syngeneic mouse model. Enhanced expression of PDL1 mediated by HDAC inhibitors was found to result from enhanced histone acetylation at the PDL1 gene promoter region. Combination therapy of HDAC inhibition and PD1 blockade was explored in the tumor setting, leading to synergistic effects in terms of reducing melanoma progression and increasing survival of B16F10 melanoma-bearing mice. These data provide a clinical rationale for combination therapy of epigenetic modifiers (e.g. HDAC inhibitors) and PD1 blockade as means to augment cancer immunotherapy, improving patient outcomes. As a second pillar of this research, the impacts of HDAC-selective inhibition were explored on immune cell biology, since the broad nature of pan-HDAC inhibitors was shown to be detrimental to T-cells in vitro and in vivo. Based on screening assay results, novel implications of treating melanoma patient T-cells ex vivo with the HDAC6-selective inhibitor ACY1215 were investigated. Treatment with this compound was unique among pan- and isotype-selective HDAC inhibitors in modulating T-cell cytokine production and showing minimal impact of T-cell viability. ACY1215 tempered Th2 cytokine production (i.e. IL-4, IL-6 and IL-10), and maintained Th1 effector cytokines (e.g. IFNγ and IL-2). Furthermore, ACY1215 increased expression of surface markers, including CD69 activation marker and ICOS co-stimulatory molecule. In addition, ACY1215 treatment enhanced accumulation of central memory T-cells during ex vivo expansion of tumor infiltrating T-cells harvested from resected tumors of metastatic melanoma patients. Importantly, ACY1215-mediated inhibition improved tumor-killing capacity of T-cells. These results highlight an unexplored ability of selective HDAC inhibitor ACY1215 to augment T-cell expansion during protocols of adoptive cell therapy. While the discoveries presented here warrant further investigation of cellular and molecular mechanisms associated with ACY1215-treated T-cells, the clinic implications are clear and rapidly translatable.

L’instabilité des microsatellites (MSI) est un phénotype tumoral dû à une déficience héréditaire ou acquise du système de réparation des mésappariements de l’ADN (MMR : mismatch repair). Le phénotype MSI est retrouvé dans 5% des cancers colorectaux métastatiques (CCRm) et est un facteur prédictif positif majeur de l’efficacité des inhibiteurs de checkpoints immunitaires (ICKi). L’objectif de mon travail a été de caractériser sur le plan clinique et moléculaire les CCRm MSI, d’en évaluer les modalités diagnostiques et d’évaluer la réponse aux ICKi. Dans un 1er travail, je montre que l’histoire naturelle clinique des CCRm MSI diffère selon le mécanisme sporadique ou héréditaire de la déficience MMR (Cohen et al., Eur J Cancer 2017). Dans un 2e travail, je montre que près de 10% des CCRm détectés comme MSI/MMR-déficients en vie réelle correspondent à des faux positifs des analyses immunohistochimiques et/ou de PCR, et que ces faux positifs sont responsables de la majorité des cas de résistance primaire aux ICKi observés dans les essais cliniques (Cohen*, Hain* et al., JAMA Oncol. 2018). Après une revue de la littérature concernant le phénotype MSI, son impact dans le cadre du CCR et des ICKi, je présente les résultats des travaux développés durant ce doctorat, avant de proposer différentes perspectives à l’ère de l’immunothérapie des cancers MSI
Microsatellite instability (MSI) is a tumor phenotype linked to somatic or germline inactivating alterations of DNA mismatch repair (MMR) genes. MSI is observed in approximately 5% of metastatic colorectal cancers (mCRC) and has recently emerged as a major positive predictive biomarker for the efficacy of immune checkpoint inhibitors (ICKi) amongst mCRC patients. The objectives of my work was to clinically and molecularly characterize MSI mCRC, to evaluate the accuracy of MSI screening methods and the response to immunotherapy in the context of ICKi clinical trials. Fist, I show that sporadic and inherited MSI mCRC display distinct natural history (Cohen et al., Eur J Cancer 2017). In a second work, I show that MSI testing in routine practice is associated with almost 10% of false positives due to misinterpration of IHC and PCR assays. Moreover, these false-positives are the main cause of mCRC primary resistance to ICKi observed in clinical trials (Cohen*, Hain* et al., JAMA Oncol. 2018). After summarizing the literature concerning MSI, its consequences on CRC and immunotherapy, I present the results of the nosologic and diagnostic works developed during this doctoral thesis. Then I will go on perspectives in the context of MSI cancer

Les mélanomes muqueux sont rares avec un potentiel métastatique important. L’immunothérapie est un traitement prometteur dans ce sous-type agressif de mélanome. L’analyse de biomarqueurs de réponse à une immunothérapie anti-PD1 chez 23 patients présentant un mélanome muqueux non opérable et/ou métastatique a montré que l’activation du complexe d’initiation de la traduction, eIF4F, était fortement prédictive de cette réponse. Cette activation a été mesurée par un test de proximité entre les sous-unités eIF4E et eIF4G. Les autres marqueurs admis, tels que l’expression tumorale de PD-L1 ou les caractéristiques de l’infiltrat lymphocytaire intra-tumoral, n’ont pas de valeur pronostique significative dans cette cohorte.La glycolyse anaérobie est la voie métabolique que la plupart des cellules tumorales privilégient lors de lacancérogenèse. Ce phénomène est appelé « effet Warburg ». Au cours de la mélanomagénèse, il existe un continuum entre l’expression croissante de l’hexokinase 2 (ou HK2, première enzyme de la glycolyse) et l’agressivité tumorale. Dans cette étude, nous avons montré que l’inhibition d’expression d’HK2 (par siARN) induisait, in vitro, une diminution majeure de la migration et de l’invasion tumorales indépendamment du métabolisme glucidique ou de l’expression initiale d’HK2. Par une technique de profilage des polysomes, nous avons observé que HK2 régulait la traduction de l’ARNm de SOX10, un facteur de transcription impliqué dans l’initiation et la progression du mélanome. Nous avons alors réalisé une immunoprécipitation de l’ARN après induction de liaisons protéine-ARN par du formaldéhyde, nous permettant ainsi de démontrer que la protéine HK2 se liait à l’ARNm de SOX10
Mucosal melanoma is a rare tumor with a high metastatic potential. Immunotherapy has promising results in this aggressive subtype of melanoma. In a cohort of 23 patients with non-resectable and/or metastatic mucosal melanoma who received anti-PD1 immunotherapy, we showed that the activation of the eukaryotic translation initiation complex, eIF4F, was a strong prognostic biomarker of response. This activation was assessed with a proximity ligation assay between eIF4E and eIF4G. The other biomarkers, such as the PD-L1 tumoral expression or the characteristics of tumor-infiltrating lymphocytes, had no prognostic value in this cohort.Aerobic glycolysis is usually the main metabolic pathway in tumor cells during cancerogenesis. This specificprocess is called “Warburg effect”. During melanomagenesis, we observed a positive correlation between the expression level of hexokinase 2 (HK2, first enzyme of glycolysis) and the tumor invasiveness. In this study, we showed that inhibition of HK2 expression (by siRNA) induced, in vitro, a major decrease of migration and invasion potential independently of the basal glycolytic metabolism or HK2 expression level in the cell lines. Moreover, performing a polysome profiling analysis, we demonstrated that HK2 was regulating the translation of SOX10 mRNA, a transcription factor involved in initiation and progression of melanoma. We, then, realized an RNA immunoprecipitation in formaldehyde and showed that HK2 was an RNA-binding protein, able to interact with the SOX10 mRNA

O cancro gástrico (GC) é o quinto cancro mais incidente e o quarto mais mortal em todo o mundo. O CG é uma doença heterogénea do ponto de vista histológico e molecular. Esta neoplasia é diagnosticada principalmente em estádios avançados da doença, onde as intervenções terapêuticas disponíveis não são eficazes. O aparecimento da imunoterapia transformou o panorama do tratamento do cancro, incluindo do GC e, atualmente, inibidores dos checkpoints imunológicos foram aprovados para o tratamento de doentes com GC recorrente ou metastático. Esta revisão resume os principais ensaios clínicos que avaliam o uso de inibidores dos checkpoints imunológicos em GC. Neste artigo destaca-se ainda o potencial da expressão de PD-L1 e da carga mutacional tumoral, bem como de aspetos característicos da classificação molecular do GC, como a instabilidade de microssatélites e a infeção pelo vírus Epstein-Barr, como biomarcadores preditivos da resposta terapêutica ao bloqueio do eixo PD-1 / PD-L1 no GC.
Gastric cancer (GC) is the fifth most incident and the fourth deadliest cancer worldwide. GC is a heterogeneous disease from the histological and molecular standpoints. This malignancy is mostly diagnosed at advanced stages of the disease, where the available therapeutic interventions are not effective. The emergence of immunotherapy has transformed the landscape of cancer treatment, including GC, and currently immune checkpoint inhibitors have been approved for the treatment of patients with recurrent/metastatic GC. This review summarizes the main clinical trials evaluating the use of immune checkpoint inhibitors in GC. It also highlights the potential of biomarkers for patient selection for GC immune checkpoint inhibition therapy, including PD-L1 expression and tumor mutational burden, and characteristics of the GC molecular classification, such as microsatellite instability status and Epstein-Barr virus infection, as predictors of response to blockade of the PD-1/PD-L1 axis.

O cancro gástrico (GC) é o quinto cancro mais incidente e o quarto mais mortal em todo o mundo. O CG é uma doença heterogénea do ponto de vista histológico e molecular. Esta neoplasia é diagnosticada principalmente em estádios avançados da doença, onde as intervenções terapêuticas disponíveis não são eficazes. O aparecimento da imunoterapia transformou o panorama do tratamento do cancro, incluindo do GC e, atualmente, inibidores dos checkpoints imunológicos foram aprovados para o tratamento de doentes com GC recorrente ou metastático. Esta revisão resume os principais ensaios clínicos que avaliam o uso de inibidores dos checkpoints imunológicos em GC. Neste artigo destaca-se ainda o potencial da expressão de PD-L1 e da carga mutacional tumoral, bem como de aspetos característicos da classificação molecular do GC, como a instabilidade de microssatélites e a infeção pelo vírus Epstein-Barr, como biomarcadores preditivos da resposta terapêutica ao bloqueio do eixo PD-1 / PD-L1 no GC.
Gastric cancer (GC) is the fifth most incident and the fourth deadliest cancer worldwide. GC is a heterogeneous disease from the histological and molecular standpoints. This malignancy is mostly diagnosed at advanced stages of the disease, where the available therapeutic interventions are not effective. The emergence of immunotherapy has transformed the landscape of cancer treatment, including GC, and currently immune checkpoint inhibitors have been approved for the treatment of patients with recurrent/metastatic GC. This review summarizes the main clinical trials evaluating the use of immune checkpoint inhibitors in GC. It also highlights the potential of biomarkers for patient selection for GC immune checkpoint inhibition therapy, including PD-L1 expression and tumor mutational burden, and characteristics of the GC molecular classification, such as microsatellite instability status and Epstein-Barr virus infection, as predictors of response to blockade of the PD-1/PD-L1 axis.

Glioblastoma is the most common and aggressive malignant brain tumor among all primary brain and central nervous system (CNS) tumors. The median survival time for glioblastoma patients given the current standard of care treatment (surgery, radiation, and chemotherapy) is less than 15 months. Medulloblastoma is another major malignant brain tumor that most frequently occurs in children. Although recent advances in surgery, radiotherapy, and chemotherapy have led to an increase in 5-year survival rates of medulloblastoma patients, treatment-related toxicity often has a major impact on long-term quality of survival.

As a result, there is an urgent need to develop more efficient and novel therapeutic approaches that specifically target tumor cells while preserving the surrounding normal CNS to improve the poor survival and quality of life of patients with malignant brain tumors. To address this need, we have developed two novel targeted immunotoxins (ITs), D2C7-(scdsFv)-PE38KDEL (D2C7-IT) and NZ-1-(scdsFv)-PE38KDEL (NZ-1-IT). D2C7-IT was developed by fusing the single-chain variable fragment (scFv) of the D2C7 monoclonal antibody (mAb) with domains II and III of Pseudomonas exotoxin A (PE38KDEL), and NZ-1-IT was developed by fusing the scFv of the NZ-1 mAb with PE38KDEL. D2C7-IT reacts with both the wild-type epidermal growth factor receptor (EGFRwt) and the EGFR variant III (EGFRvIII), two overexpressed proteins in glioblastomas. NZ-1-IT reacts with podoplanin (PDPN), a protein that has a high expression in glioblastomas and medulloblastomas.

In vitro cytotoxicity data shows that both ITs effectively inhibited protein synthesis in a variety of epitope-expressing glioblastoma and medulloblastoma xenograft cells and human tumor cell lines. Furthermore, the direct anti-tumor efficacy of D2C7-IT was examined in orthotopic glioma models in immunocompromised mice, while the direct anti-tumor efficacy of NZ-1-IT was observed in medulloblastoma xenograft-bearing immunocompromised mice. Both immunotoxins showed a robust anti-tumor efficacy in the preclinical brain tumor models. D2C7-IT was first investigated in the subsequent studies to accelerate its translation to the clinic. The preclinical toxicity of intracerebral D2C7-IT infusion was subsequently determined in normal Sprague-Dawley (SD) rats. The maximum tolerated dose (MTD) of D2C7-IT was determined to be between a total dose of 0.10 and 0.35 μg, and the no-observed-adverse-effect level (NOAEL) of D2C7-IT was a total dose of 0.05 μg in SD rats. Both the MTD and NOAEL were utilized as references for the D2C7-IT clinical trial design.

In addition to direct tumor cell killing, immunotoxin monotherapy has been shown to induce a secondary anti-tumor immune response through the engagement of T cells. Therefore, the D2C7-IT-induced secondary anti-tumor immune response was investigated using syngeneic mouse glioma models in immunocompetent mice. Moreover, previous studies have demonstrated that immune checkpoint inhibitors have a robust anti-tumor efficacy by augmenting the T cell response to the tumor cells. Thus, immune checkpoint inhibitors were combined with D2C7-IT in order to enhance the immunotoxin-induced anti-tumor immune response to eliminate residual tumor cells and prevent tumor recurrence in the long term. Meanwhile, studies with NZ-1-IT remain preliminary; thus, this IT will not be as robustly discussed as D2C7-IT throughout this text.

Dissertation

Research Doctorate - Doctor of Philosophy (PhD)
Malignant melanoma is one of the most aggressive and lethal cancers of the skin. Clinical reports have shown patients diagnosed with stage IV of the disease have no longer than five years to live. Depending on the sub-stage of the disease, median survival of patients is between 6 - 18 months. To date, current Food and Drug Administration (FDA) approved anticancer drugs for melanoma have demonstrated limited response rates, few complete remissions and no significant survival benefits. The development of new therapies to effectively eradicate malignant melanoma are desperately in need. Recent growth in the field of oncolytic virotherapy has led to an increased number of clinical trials and acceptance of tumour selective viruses as a promising anticancer strategy. Numerous viruses have emerged as potent oncolytic agents because of their capacity to preferentially infect and destroy cancer cells while leaving normal cells intact. Like most anticancer modalities, it is very likely that oncolytic virotherapy will be used in combination with other existing therapeutics. Numerous groups have begun to explore the possibility of combining oncolytic virotherapy with conventional cancer therapies. The combination of oncolytic virotherapy with other existing cancer therapies may be an effective strategy to overcome the barriers faced by either therapeutic agents when used as monotherapies. The Kuykendall strain of Coxsackievirus A21 (CVA21) is a naturally occurring common-cold virus that has the inherent capacity to preferentially infect and destroy malignant cells. The oncolytic properties of CVA21 as an anticancer agent have been demonstrated both in vitro and in vivo studies, against numerous types of cancer, including breast cancer, prostate cancer, glioma, multiple myeloma, non-small cell lung carcinoma, and melanoma. In the clinic, tumour regression was observed in both injectable and distant non-injected lesion without any significant adverse events. Despite CVA21’s impressive progress to date, it is becoming clear the oncolytic viruses (OVs) cannot be viewed as monotherapies. The current trend is for the combination of virotherapy with mainstream therapies. A fundamental requirement for the success of this strategy is that the combination does not interfere with the life cycle of the oncolytic virus from the point of viral entry, to the release of viral progeny. In Chapter 4, we demonstrated that not only does the co-administration of clinically relevant cytotoxic anticancer drugs such as dacarbazine, paclitaxel and carboplatin not interfere with replication cycle of CVA21, enhanced cell destruction of melanoma cells was achieved when both oncolytic agent and chemotherapy were combined. To determine whether actual synergism was present, we subjected our data to the Chou-Talalay median effect equation which provides a robust measurement of drug combination relationships based on combination index values. When the constant drug-ratio design was applied to our combination assays, we discovered that CVA21 does indeed act synergistically with conventional chemotherapy in all melanoma cell lines except SK-Mel-28. However, we anticipate that it is possible to achieve synergistic or additive effects in all cell lines by carefully adjusting the drug concentrations used. Indeed, the combination of CVA21 with either of the chemotherapeutic agents at clinically relevant drug concentrations, resulted in increased cell death in SK-Mel-28 cells. Next, we were interested in identifying the underlying mechanism by which CVA21 is able to synergise with dacarbazine, paclitaxel and carboplatin. No increase in viral replication was observed in any of the test cell lines following combination chemotherapy treatment vs virus alone. Cell cycle analysis revealed an increase in the sub-G1/G₀ cell population after treatment with the combination of CVA21 and paclitaxel + carboplatin, but was not associated with the activation of caspase-3/7. Taken together, the combination of CVA21 plus chemotherapy has significant activity in a panel of melanoma cells though the mechanism behind this effect remains to be elucidated. In Chapter 5, we sought to determine whether the synergistic cancer cell death was reproducible in vivo. Two melanoma animal models were designed; an immunodeficient melanoma xenograft model and a syngeneic immunocompetent mouse model. Intratumoural injection of infectious CVA21 particles in the presence or absence of cytotoxic chemotherapy mediated complete regression of all injected melanoma xenografts. However, there was no statistical difference between single agent CVA21 and CVA21 in combination with chemotherapy in our immunodeficient mouse model. Such a result was not unexpected, as the absence of neutralising antibodies in the immunodeficient model allowed the virus to replicate indefinitely until all tumour cells were destroyed. Having proven that cytotoxic chemotherapy failed to interfere with the replication cycle of CVA21 in vivo, we developed a syngeneic immunocompetent mouse model and treated the animals with the same combinations as per the immunodeficient study. Overall, we observed an excellent level of treatment tolerability and tumour clearance in both single agent/combination therapy treatment groups. Results from both animal models show that the presence of either chemotherapy does not harm the infectivity or integrity of CVA21 virions in vivo and provides a strong rationale for clinical translation. In Chapter 6, we explored the immunotherapeutic potential of oncolytic CVA21. In the first section of this chapter, we examined whether vaccination of immunocompetent animals with cell lysates induced by CVA21 oncolysis (an oncolysate) could initiate the activation of anti-tumour immunity. Following immunisation with the oncolysates, animals were challenged with B16 cells and the development of flank tumours was recorded. Although B16 challenged animals showed reduced tumour growth, it became apparent that viral-mediated lysis of tumour cells alone was insufficient for complete tumour eradication. Thus, based on these observations, the next logical step was to combine oncolytic CVA21 with other immunotherapeutic agents to enhance the activation of anti-tumour immune responses. In the next section, we investigated if the immunomodulatory effects of two T cell regulatory receptors, PD-1 and CTLA-4, together with CVA21 could be harnessed to improve therapeutic efficacy. Animals were treated with intratumoural injections of CVA21 and intraperitoneally with either anti-PD-1 or anti-CTLA-4 antibodies. Animals were rechallenged with B16 cells lacking the CVA21 targeting receptor human ICAM-1 and the development of the secondary flank tumour was used as a measure for the generation of a protective anti-tumour immune response. Remarkably, the combination group displayed a superior anti-tumour response when compared to control animals or either of the agents as monotherapies. We were particularly successful in demonstrating this effect with the CVA21-CTLA-4 combination as indicated by the the complete regression of the primary tumour in all treated animals, followed by durable resistance to the secondary tumour challenge in 40% of the animals on study. The experimental data presented herein highlights the potential of CVA21 as a useful platform for combination strategies. Using the newly established, fully immunocompetent CVA21-susceptible mouse model of malignant melanoma, we showed that CVA21 in combination with dacarbazine or paclitaxel and carboplatin had significant advantages over either agents as monotherapies. Furthermore, the model was extended to evaluate the protective anti-tumour response generated from CVA21 oncolysis by challenging animals with murine melanoma cells without ICAM-1 surface receptors after the initial treatment. Favourable immune responses and increased therapeutic benefits were observed when CVA21 was administered with the immune checkpoint inhibitors PD-1 and CTLA-4. This study is further proof-of-concept that immunovirotherapy of cancer is achievable with CVA21, leading the way for future clinical trials combining checkpoint modulation with CVA21.

Indiana University-Purdue University Indianapolis (IUPUI)
Purpose: The purpose was to comprehensively assess the impact of PD-L1 expression on the efficacy of immune checkpoint inhibitors on Overall Survival (OS) and Progression-Free Survival (PFS). Methods: A systematic literature search and review was conducted through June 2019. I searched all eligible randomized controlled trials comparing PD-1/PD-L1 monotherapy to an active comparator in adult patients with advanced cancer across multiple tumor types. The Cochrane risk-of-bias tool was used to assess trial quality. A random-effects model was used for the meta-analysis. Heterogeneity was assessed using Cochran Q statistic and I2 test. Publication bias was assessed by visual inspection of a funnel plot and Begg’s test. Results: I identified and included 23 trials involving 14,434 participants. When stratifying PD-L1 positive (+) and negative (-) patients using varying thresholds of expression, a significant group difference was observed at PD-L1 >1% ( p=0.04; PD-L1(+): HR, 0.72; 95% CI, 0.65-0.79; PD-L1(-): HR,0.83; 95% CI, 0.75-0.91), at PD-L1 >10% (p=0.02; PD-L1(+): HR,0.50; 95% CI, 0.38-0.62; PD-L1 (-): HR, 0.74; 95% CI, 0.57-0.90) and at PD-L1>50% (p=0.01; PD-L1(+): HR,0.59; 95% CI, 0.51-0.68; PD-L1(-): HR, 0.93; 95% CI, 0.71-1.15). Across tumor types, both PD-L1(+) and PD-L1(-) patients treated with an immunotherapy had improved OS compared with patients receiving standard care therapies. A PFS benefit was observed and favored patients treated with a PD-1/PD-L1 inhibitor versus standard of care. However, there was significant heterogeneity and the benefit on PFS was not statistically significant between PD-L1(+) and PD-L1(-) groups using varying cut-off levels of PD-L1 expression. No differences between sub-groups of interest including median follow-up time, type of inhibitor, and line of therapy for either PD-L1(+) or PD-L1(-) patients at 1% cut-off were identified. Conclusion: This study supports the use of PD-L1 as a predictive biomarker of improved response to immunotherapies. As thresholds increase and specifically above the 10% PD-L1 expression threshold, patients who were positive for PD-L1 appeared to have better OS compared to those who were negative for PD-L1. Further investigation is needed to assess the clinical usefulness of PD-L1 at various expression levels with improved technologies that have the potential to enhance assay accuracy and precision.

The interactions of tumours with normal host tissue are key determinants of cancer growth and progression. The ability or inability of the patient’s immune system to mount a response against the tumour is tightly correlated with prognosis. One of the ways tumours avoid detection and elimination by the immune system is by expressing programmed death ligand 1 (PD-L1). PD-L1 binds to its receptor programmed death 1 (PD1) on T cells, inhibiting T cell responsiveness to antigenic stimuli. Blockade of the PD1/PD-L1 pathway removes this negative signal and restores anti-tumour immunity. While this blockade of PD1/PD-L1 is well established through the use of antibodies, small molecule inhibitors of PD1/PD-L1 are relatively unknown. We employed in silico docking in order to find small molecules capable of binding to either PD1 or PD-L1, and the highest-ranked compounds were tested in biophysical assays for their ability to inhibit PD1/PD-L1 binding. A thermal shift assay identified a pyrazole compound as a possible binding partner for PD-L1, but follow-up assays showed that it had no effect on the PD1/PD-L1 interaction and that its apparent binding was probably due to aggregation. An ELISA assay identified a tryptophan diamine compound as an apparent stabilizer of the PD1/PD-L1 interaction. However this compound, too, was later identified to be inactive in orthogonal assays. We identified a family of salicylic acid derivatives that interfered with TR-FRET measurements – an unusual observation, given that TR-FRET is touted as being insensitive to most mechanisms of compound interference. This discovery should help other fragment- screening groups identify false positives more easily. We also probed the mechanism of inhibition of a recently disclosed family of small molecule PD1/PD-L1 inhibitors from Bristol-Myers Squibb. Concurrently with other groups, we used protein NMR, size exclusion chromatography, and SPR to determine that the compounds were inducing homodimerization through the PD1-binding face of PD-L1. Furthermore, using cellular crosslinking and live cell imaging, we showed that these first generation inhibitors are fairly ineffective at inhibiting this interaction on the cell surface. More potent compounds will be needed to see any cellular effect from this mechanism of action.
Graduate
2019-02-15

Modulation of immune modulatory pathways has emerged as one of the most successful and explored approaches for cancer immunotherapy. Current therapeutics include monoclonal antibodies, which have shown impressive clinical outcomes in the treatment of several types of tumors. However, low response rate, patient acquired resistance and induction of severe immune-related adverse effects represent critical drawbacks. To overcome these limitations, complementary strategies that inhibit tumor immunosuppressive pathways and enhance immunity are urgently needed. We hypothesized that inhibition of PD-1/PD-L1 interaction, for immunosuppression blockade, could be improved by the development of immune modulatory small molecules. For this purpose, we developed a computer-assisted drug design approach that allowed the identification of potential PD-1/PD-L1 small-molecule inhibitors. These potential hits were confirmed as true hits by in vitro and ex vivo assessment of its antitumor immune response. The compounds identified were able to activate T cell function by increasing tumor-associated antigen recognition, processing and presentation to effector T cells. The use of computational-aided drug design tools was decisive to accelerate the identification of new immune small molecules modulators which, certainly, will play a key role in the next generation of cancer immunotherapy approaches. Hits were identified by a structure based virtual screening campaign based on molecular docking. In addition to hit discovery, these methods were essential in providing a strong structural elucidation. Over biochemical and in vitro studies three PD-1/PD-L1 small-molecule inhibitors by direct PD-L1-binding were identified at nanomolar scale. Two different types of cancer, a highly immunogenic (melanoma) and a traditionally non-responder to immune checkpoint modulation (breast cancer) were studied in vitro. Finally, co-culture experiments were developed and the treatment with the identified inhibitors led to T cell activation and antigen-specific cytotoxic T-cell activity against melanoma cells ex vivo. Altogether, these findings reveal important aspects on the use of small molecules as immune checkpoint modulators, as well as their effect on the modulation of tumor-infiltrating immune cells. This approach offers numerous advantages over the use of monoclonal antibodies, predominantly on pharmacokinetics properties. In addition, they represent a more affordable and accessible alternative. Therefore, small-molecule immune system modulators emerge as a potential strategy to improve clinical outcomes of cancer treatment.

A imunoterapia contra o cancro é uma das estratégias mais recentes utilizadas para combater o cancro, que ativa ou estimula o sistema imunológico para produzir efeitos antitumorias. Diversos cancros são altamente refratários à quimioterapia convencional, o que em vários casos a sobrevivência de tumores é conseguida por imunomodelação de checkpoint para manter o desequilíbrio entre a vigilância imune e a proliferação das células cancerígenas. A imunoterapia por checkpoints inhibitors está a mudar, ao longo dos tempos, a terapêutica do cancro. Há cada vez mais indicações terapêuticas para o seu uso. Estudos clínicos mostram cada vez mais eficácia numa grande variedade de tumores sólidos e hematológicos, embora alguns ensaios clínicos também mostram resultados negativos. Os anticorpos de checkpoints inhibitors, como anti-PD-1 e anti-PD-L1, são uma nova classe de inibidores que funcionam como um fator de supressão de tumores através da modulação da interação com as células tumorais imunes. Estes bloqueadores de checkpoint estão a tornar-se numa abordagem terapêutica altamente promissora para o cancro que produz grande variedade de respostas antitumorais com efeitos colaterais reduzidos. Apesar deste enorme sucesso na eficácia da resposta à terapêutica anti-PD-1 e anti-PD-L1, é limitado a tipos específicos de tumores, devido à expressão insuficiente e heterogénea de PD-1 no microambiente tumoral. Cada vez mais existem diferentes anticorpos inibidores do PD-1e PD-L1. Existem novos conceitos para o tratamento combinado com immune checkpoint inhibitors e terapêuticas convencionais, e por isso estes regimes de combinação devem ser desenvolvidos e explorados uma vez que nem todos os pacientes respondem da mesma forma à imunoterapia. Também existe a necessidade de melhorar os biomarcadores para identificar os pacientes que beneficiarão mais do tratamento por immune checkpoint inhibitors ou terapêuticas combinadas.
Immunotherapy against cancer is one of the most recent strategies used to combat cancer, which activates or stimulates the immune system to produce antitumor effects. Several cancers are highly refractory to conventional chemotherapy, which in many cases tumor survival is achieved by immunomodeling a checkpoint to maintain the imbalance between immune surveillance and cancer cell proliferation. Immunotherapy by checkpoints inhibitors is changing, over time, cancer therapy. There are more and more therapeutic indications for its use. Clinical studies are increasingly effective on a wide range of solid and hematological tumors, although some clinical trials also show negative results. Checkpoint inhibitor antibodies, such as anti-PD-1 and anti-PD-L1, are a new class of inhibitors that works as a tumor suppressor factor by modulating the interaction with immune tumor cells. These checkpoint blockers are becoming a highly promising therapeutic approach to cancer that produces a wide variety of antitumor responses with reduced side effects. Despite this enormous success in the efficacy of the response to anti-PD-1 and anti-PD-L1 therapy, it is limited to specific types of tumors, due to the insufficient and heterogeneous expression of PD-1 in the tumor microenvironment. Increasingly there are different inhibitors of PD-1 and PD-L1. There are new concepts for combined treatment with immune checkpoint inhibitors and conventional therapies, and therefore these combination regimens should be developed and explored since not all patients respond in the same way to immunotherapy. There is also a need to improve biomarkers to identify patients who will benefit most from treatment by immune checkpoint inhibitors or combination therapies.

abstract: Immunotherapy has received great attention recently, as it has become a powerful tool in fighting certain types of cancer. Immunotherapeutic drugs strengthen the immune system's natural ability to identify and eradicate cancer cells. This work focuses on immune checkpoint inhibitor and oncolytic virus therapies. Immune checkpoint inhibitors act as blocking mechanisms against the binding partner proteins, enabling T-cell activation and stimulation of the immune response. Oncolytic virus therapy utilizes genetically engineered viruses that kill cancer cells upon lysing. To elucidate the interactions between a growing tumor and the employed drugs, mathematical modeling has proven instrumental. This dissertation introduces and analyzes three different ordinary differential equation models to investigate tumor immunotherapy dynamics. The first model considers a monotherapy employing the immune checkpoint inhibitor anti-PD-1. The dynamics both with and without anti-PD-1 are studied, and mathematical analysis is performed in the case when no anti-PD-1 is administrated. Simulations are carried out to explore the effects of continuous treatment versus intermittent treatment. The outcome of the simulations does not demonstrate elimination of the tumor, suggesting the need for a combination type of treatment. An extension of the aforementioned model is deployed to investigate the pairing of an immune checkpoint inhibitor anti-PD-L1 with an immunostimulant NHS-muIL12. Additionally, a generic drug-free model is developed to explore the dynamics of both exponential and logistic tumor growth functions. Experimental data are used for model fitting and parameter estimation in the monotherapy cases. The model is utilized to predict the outcome of combination therapy, and reveals a synergistic effect: Compared to the monotherapy case, only one-third of the dosage can successfully control the tumor in the combination case. Finally, the treatment impact of oncolytic virus therapy in a previously developed and fit model is explored. To determine if one can trust the predictive abilities of the model, a practical identifiability analysis is performed. Particularly, the profile likelihood curves demonstrate practical unidentifiability, when all parameters are simultaneously fit. This observation poses concerns about the predictive abilities of the model. Further investigation showed that if half of the model parameters can be measured through biological experimentation, practical identifiability is achieved.
Dissertation/Thesis
Doctoral Dissertation Applied Mathematics 2020

Head and neck squamous cell carcinoma encompasses a complex and heterogeneous group of malignant diseases. Originally, this tumor type was associated with tobacco and alcohol consumption. However, a significantly expanding subset of tumors associated with oncogenic human papillomavirus infection arising in deep tonsillar crypts was identified within the last decades. Due to the essential role of the immune system in antiviral and anticancer immune response, the prognosis of patients is significantly influenced by the volume, composition and functional capacity of the immune infiltrate. The immunosuppressive landscape of head and neck cancer leads to unfavorable outcome of patients and decreased efficacy of immunotherapy. The response rate to standard treatment is high, however, standard therapy is accompanied by considerable toxicity influencing the quality of life. In 2016, the first immunotherapeutics for the treatment of patients with recurrent squamous cell carcinoma of the head and neck were approved - the anti-PD-1 immune checkpoint inhibitors nivolumab and pembrolizumab. This type of therapy, based on mitigation of immunosuppression, shows strong efficacy and less toxicity in combination with other therapies. Therefore, anti-PD-1 immunotherapy was recently approved in the first-line...