Research Sébastien Anguille

Abstract

Acute myeloid leukemia (AML) treatment remains challenging despite therapeutic advances. About 50% of patients in complete remission (CR) will relapse within 5 years after initial diagnosis. In the past decade, chimeric antigen receptor (CAR)-T cell therapy has been very successful in hematological malignancies like B-cell lymphoma. However, in AML, results have been highly unsatisfactory, despite several preclinical and clinical CAR-T studies. Challenges in CAR-T cell therapy for AML include AML heterogeneity, lack of a leukemia-specific antigens leading to off-tumor toxicity, and an immunosuppressive bone marrow (BM) microenvironment. In this PhD project, I aim to investigate and tackle these main problems in AML CAR-T treatment by developing CAR-T cells using BM-derived tumor infiltrating lymphocytes (TILs) as a novel CAR-T-cell source and a logic AND-gated approach with co-stimulatory receptors (CCR) and signaling CARs, generating AND-gated BM-derived CCR+CAR-engineered TILs. For this, I will (1) find a suitable protocol for BM-derived TIL expansion, (2) profile BM-derived TILs of AML patients, (3) find a combination of antigens more specific for AML and not for normal HSCs, using logic AND-gated CAR strategy, and (4) evaluate the antileukemic functionality of AND-gated CCR+CAR-BM-TILs. This project will bring closer a new and potent generation of CAR-T therapies for leukemia.

Researcher(s)

• Promoter: Anguille Sébastien
• Co-promoter: Campillo Davó Diana
• Fellow: Katana Zoi

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of certain hematologic cancers. In this immunotherapy, the patient's T-cells are "armoured" ex vivo with a CAR that targets certain antigens on the tumor cell surface. Once administered, the CAR-T-cells will recognize the tumor cells and mediate lysis of the cells However, CAR-T-cell therapy is not yet a breakthrough for acute myeloid leukemia (AML), a highly aggressive blood cancer with dismal prognosis, due to various reasons. One of the reasons is the lack of a suitable CAR-target antigen on the AML cell surface. Another contributing factor is that the T-cells in AML, which are usually taken from peripheral blood, are deemed suboptimal. It is possible that tumor-infiltrating lymphocytes (or TIL) represent a superior cell population, but little research in the CAR therapy field is focused on TIL. To our knowledge, no research has been conducted on the use of TIL for the development of CAR-T cell therapy in AML. The aim of this project is twofold, with the ultimate goal of developing a new CAR-T-cell therapy for AML. Firstly, in this project, a new CAR targeting a promising target antigen that is highly expressed on the AML cell surface will be tested. Secondly, we wish to determine whether bone marrow-derived TIL may be suitable as a source for CAR-based therapy for AML. In a model of AML, we will perform an extensive phenotypic, transcriptional, and functional characterization of anti-AML CAR-engineered TIL compared to conventional peripheral blood lymphocytes.

Researcher(s)

• Promoter: Anguille Sébastien
• Fellow: Katana Zoi

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Multiple myeloma is a rare form of white blood cell cancer of the bone marrow. While there is no cure, multiple myeloma can be managed successfully in many patients for years because of the growing availability of new drugs. Despite these advances, most current treatment strategies follow a one-size-fits-all approach and novel techniques to select patients for specific therapies are needed, especially considering the potential toxicity and cost of emerging immunotherapies (like CAR-based cellular therapies). Moreover, pockets of myeloma cells can exist within a patient with different sensitivity for a specific treatment, and single-site bone marrow biopsy may be less reliable to identify heterogeneous disease. Positron emission tomography (PET) provides a powerful platform to characterize tumors non-invasively by modifying and radio-labeling antibodies to image the tumor phenotype. In this preclinical project, we will develop, validate, and assess the predictive potential of a new antibody-based PET tracer to assess BCMA, a protein that is highly and selectively expressed on myeloma cells, offering our radiopharmaceutical unique specificity. Finally, a mouse model expressing human characteristics will be used to assess our tracer in a clinically relevant setting using CAR-based cell therapy. If successful, our tracer will help physicians select patients who can benefit from CAR therapy and avoid risking the severe side-effects in patients with a low likelihood of response.

Researcher(s)

• Promoter: Anguille Sébastien
• Co-promoter: Elvas Filipe
• Co-promoter: Van den Wyngaert Tim

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Hematologic malignancies are cancers that primarily affect the blood, bone marrow and lymph nodes. Among the different subtypes, B-cell non-Hodgkin lymphoma (B-NHL) and Acute Myeloid Leukemia (AML) are the most prevalent indications. Common to both indications, alterations in the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway are frequently observed, leading to constitutive activation and oncogenic signalling. Known key effectors within the pathway such as Bruton's tyrosine kinase (BTK), interleukin-1 receptor associated kinases (IRAKs), Myeloid differentiation primary response 88 (MYD88), and Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) represent promising therapeutic targets for these indications and have been at the center of significant drug discovery efforts. In order to tackle common limitations associated to canonical small molecule inhibitors (SMI) (e.g., resistance mutations, lack of response due to scaffolding functions, …), this project is aimed at exploring the therapeutic potential of selective target degradation through PROteolysis-TArgeting Chimera (PROTAC). PROTAC represents an innovative protein degradation technology able to induce protein degradation by taking advantage of the ubiquitin proteasome pathway. The goal of the project is to provide a better understanding of the therapeutic potential of PROTACs specific for BTK, IRAK1 and IRAK4 in comparison to their respective SMI counterparts. To this end, we will evaluate the molecular and functional consequence of target degradation or inhibition in relevant models of AML and B-NHL as well as reflect the work on AML primary patient material. In addition, potential synergistic activity between PROTACs and clinically relevant SOC/SMIs options will be evaluated. Last, potential on-target/off-tumor activity in immune cell sub-types in which the NF-kB pathway is known to play a role (e.g., T-cells) will be evaluated.

Researcher(s)

• Promoter: Lion Eva
• Co-promoter: Anguille Sébastien
• Fellow: Suls Toon

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Flow cytometry is a widely used technique that allows the simultaneous and multi-parameter analysis of physical and biochemical characteristics of a population of living cells or particles in a heterogenous sample. The Laboratory of Experimental Hematology (LEH) has 20+ years of demonstrable experience in flow cytometry (200+ published manuscripts), as well as experience in guidance and support of both internal and external research groups with flow cytometric experiments (30+ joint manuscripts). With this application, we now aim to maintain and expand a flow cytometry and cell sorting core facility at the AUHA. The ambition of LEH is to make basic and advanced flow cytometry available for all active and prospective users at the AUHA in order: (i) to leverage qualitative cell biological and (pre)clinical cellular research, (ii) to provide qualitative education covering flow cytometry and its applications over multiple faculties (FGGW, FBD and FWET), and (iii) to provide external service using flow cytometry as a basis, both intellectually as well as practically.

Researcher(s)

• Promoter: Cools Nathalie
• Co-promoter: Anguille Sébastien
• Co-promoter: Lion Eva
• Co-promoter: Ponsaerts Peter

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Most CAR-T cell therapies for treatment of multiple myeloma (MM) are directed towards BCMA, a target antigen that is highly expressed on malignant plasma cells. It has become evident that the extracellular BCMA-binding domain of a CAR, derived from a monoclonal antibody (mAb), is an important determinant of clinical efficacy of anti-BCMA CAR-T cell therapies. In this project, we want to further investigate and compare anti-BCMA mAbs from different animal species for their competence for incorporation in CAR-T cell therapies against MM. In addition, most CAR-T cells get their genetic material delivered via viral transduction or transposons. A major disadvantage of viral loading methods are the need for highly specialized infrastructure and the long time needed for production. The speed up the production process, make it safer and to counteract virus-mediated insertion of DNA in the genome, we want to use episomal vectors. These vectors exist extrachromosomal, yet are duplicated and thus passed along to daughter cells. Next, we want to turn on/off specific CARs by (de)methylating the genes, thereby regulating transcription. It also enables speeding up and optimization of the production process by incorporating different CARs in a single T-cell, whereby only the CAR of choice is switched on. This could be incorporated into allogenic cell therapy in which different CARs are present in an off-the-shelf allogenic T-cell/NK-cell.

Researcher(s)

• Promoter: Anguille Sébastien
• Fellow: Shaw Philip

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Confidential .

Researcher(s)

• Promoter: Lion Eva
• Co-promoter: Anguille Sébastien
• Co-promoter: Berneman Zwi

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Chimeric antigen receptor (CAR)-T cell therapy is an innovative form of cellular immunotherapy that utilizes T-lymphocytes that are genetically engineered to express a CAR. While initial response rates are often outstanding, the majority of patients suffers from a relapse which is often cause by a lack of sustained effector functionality or persistence of the CAR-T cells. The importance of the CAR architecture to therapeutic outcome is becoming increasingly clear. However, it is unlikely that the full potential of CAR-T-cell therapy will be reached by using a combination of domains derived from only a small subset of immune-related proteins, as is the case today. The main reason behind this limited selection of building blocks is the use of slow and labor-intensive low-throughput methods for the evaluation of novel candidate domains. Only few groups, such as the Birnbaum lab, have developed a high-throughput workflow for the functional evaluation of up to 1 million CAR designs. While those workflows provide invaluable information novel costimulatory domain combinations, it has never been applied to other structural domains of CARs. The aim of this research stay at the Birnbaum lab is to acquire the practical know-how on CAR combinatorial library construction and a high-throughput CAR screening workflow. By applying this knowledge directly under the supervision of the host group, we will attempt to decipher the design language of severely understudied CAR components by screening a sizeable library of potentially valuable CAR domains.

Researcher(s)

• Promoter: Anguille Sébastien

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Genetic engineering of lymphocytes for adoptive cell transfer has marked a turning point in personalized immunotherapy, especially in the treatment of cancer. Adoptive T-cell immunotherapies using antitumor chimeric antigen receptors (CARs) and T-cell receptors (TCRs) have, however, not met expectations yet for the majority of malignancies, including acute myeloid leukemia (AML). Moreover, expression of immunosuppressive immune checkpoints (IICPs) hinders the success of these therapies. To address the shortcomings of current T-cell therapies, the aim of this research project is to develop an innovative combinatorial and genetically engineered adoptive T-cell therapy focusing on AML as a disease model. In this project four important issues will be covered. First, cancer cells capitalize on processes such as downregulating peptide-major histocompatibility complex (pMHC) ligands to lower their immunogenicity and, by doing so, evade immune detection. Second, TCRs that target tumor self-antigens are scarce and usually have low affinities, having difficulties in binding target tumor antigens. Finding ways to improve interaction between pMHC ligands and low affinity TCRs, such as those that target self-antigens, would improve the chance of success in TCR-engineered T-cell therapies. Third, adoptive T-cell therapies are confronted with immunosuppressive environments that hinder their efficacy via engagement of IICPs, such as PD-1, TIM-3, or LAG-3. Determining the most relevant IICPs is key for developing effective adoptive T-cell therapies. Fourth, these therapies must be tumor-specific and efficacious once translated into a clinical setting. Taken together, combinatorial and flexible approaches for TCR-engineering will mark the next-generation of T-cell immunotherapies, by addressing (a) improved interaction between T cells and cancer cells, (b) immune evasion through IICPs, (c) cost-effectiveness of an all-in-one therapy, and (d) safety using RNA-based methods. In summary, improved adoptive T-cell therapies that overcome CAR and TCR challenges as well as the immunosuppressive environment that hinders antileukemic T-cell action will facilitate innovative solutions for cancer treatment.

Researcher(s)

• Promoter: Lion Eva
• Co-promoter: Anguille Sébastien
• Fellow: Flumens Donovan

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project website

Project type(s)

• Research Project

Abstract

Allogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment modality for patients with relapsed/refractory (r/r) CD19+ B-cell hematological malignancies, such as acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL). The prognosis of patients in whom the disease is not under control or has relapsed after HSCT, is particularly grim. For these patients, the therapeutic options are limited. One of the few available salvage strategies involves the use of donor lymphocyte infusions (DLI). The mechanism of action of DLI relies on the administration of immune effector cells, predominantly T cells, from the stem cell donor, with the ultimate goal to elicit a "graft-versus-leukemia" or "graft-versus-tumor" effect. Unfortunately, DLI have only modest clinical activity and can evoke or exacerbate serious transplant-related side effects such as "graft-versus-host" disease. CD19-targeted chimeric antigen receptor (CAR)-T cell therapy offers new hope for patients with r/r B-cell hematological malignancies. Here, T-cells derived from the patient (autologous) are genetically modified to express a CD19 CAR, a synthetic receptor enabling binding of the cells to the CD19-expressing target cells. Upon engagement, the CAR will trigger activation of the T cells which will then become cytotoxic towards the target cells. In this project, DLI products will be loaded with an in-house developed and optimized CD19 CAR. By using allogeneic cells derived from healthy donors as source for CAR-T cell manufacturing, which are usually "fitter" than autologous, patient-derived T cells, we aim to enhance the anti-tumoral activity of the CAR-T cells.

Researcher(s)

• Promoter: Anguille Sébastien
• Fellow: De Laere Maxime
• Fellow: Krekelbergh Laurens
• Fellow: Krekelbergh Laurens

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Acute myeloid leukemia (AML) is a rare type of cancer that predominantly affects people in the third age. The 5‐year overall survival rate of AML patients is only 30%, a figure that has not substantially changed despite enormous therapeutic advances in the last decade. Novel immunotherapies, such as T-cell receptor (TCR) T-cell and chimeric antigen receptor (CAR) T-cell therapies, are difficult to adopt in the context of AML. This is because most AML-related antigens are intracellular self-antigens that are expressed on the AML cell surface as peptides via major histocompatibility complexes (MHC); TCRs specific for these self-antigens are difficult to obtain since self-reactive T cells undergo thymic negative selection. In contrast to CD19 which is a very suitable extracellular target antigen for CAR-T cell therapy in acute lymphoblastic leukemia (ALL), the very few extracellular antigens expressed on AML cells that can serve as targets for CAR-T cell-based therapies, such as CD33 and CD123, are also expressed on normal hematopoietic stem/progenitor cells entailing a risk of intolerable myeloablation. The aim of this innovative project is to combine the best of two worlds, namely to redirect T-cells towards the key intracellular AML antigen Wilms' tumor protein 1 (WT1) using WT1-specific TCRs, combined with an innovative non-signaling CAR (NSCAR) towards a novel candidate extracellular AML antigen.

Researcher(s)

• Promoter: Lion Eva
• Co-promoter: Anguille Sébastien
• Co-promoter: Campillo Davó Diana

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Chimeric antigen receptor (CAR)-T cell therapy has demonstrated unprecedented clinical activity in patients with hematological diseases, but a large proportion of them will ultimately relapse. Further optimization of this new treatment modality is therefore required to unlock its full therapeutic potential. In this project, in addition to using readily available cell line models, we will use our mRNA electroporation technology for CAR loading of immune cells. This will provide a rapid and efficient way to explore new research paths that can lead to optimized CAR-based cellular therapies for hematological diseases. Will assess the value of a multi-targeted approach incorporating two established CAR targets (CD19 and B-cell maturation antigen) and the novel CAR candidate CD200. Next, the hinge and co-stimulatory domains in the CAR structures will be sequentially modified, comparing conventional hinge and co-stimulatory domains with our recently discovered 4-1BB-hinge and CD26 co-stimulatory domains. Exhaustion will be prevented by introducing programmed death (PD-1) silencing RNA in the CAR-modified cells to reduce PD-1-mediated co-inhibitory signaling. Finally, positive findings will be translated from our cell line models to conventional T cells, NK cells and gdT cells.

Researcher(s)

• Promoter: Anguille Sébastien
• Co-promoter: Lion Eva
• Fellow: Roex Gils

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

The field of chimeric antigen receptor (CAR)-T-cell immunotherapy has evolved tremendously over the past decades. One of the milestones in CAR development was the incorporation of co-stimulatory domains, providing the necessary signaling to trigger full T-cell activation. Given their improved performance, these so-called second-generation CAR-T cells equiped with CD28- or 4-1BB -based intracellular domains dominate the clinical trial landscape. For multiple myeloma, the second most common type of blood cancer, early-phase clinical trials with B-cell maturation antigen (BCMA)-targeted CAR-T cells have demonstrated promising results, but relapses are frequently observed. Therefore, a great deal of research attention is currently being paid at improving the efficacy of the CAR-T cells. In the proposed project, we want to evaluate the potential of CD26 as a co-stimulatory domain in BCMA-targeted CARs. CD26 has been shown to improve persistence and anti-tumor activity of T cells, and is associated with a memory phenotype. In addition, the impact of the hinge domain on CAR-T-cell efficacy has been poorly studied, although the hinge domain has been shown to be important for antigen recognition. Therefore, we developed a novel 4-1BB-based hinge in collaboration with colleagues at Fudan University, Shanghai, China. This new hinge was already validated in the context of anti-HER-2, -GPC3 and -CD19 CAR-T cells, and will be further evaluated here in multiple myeloma.

Researcher(s)

• Promoter: Anguille Sébastien

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Acute myeloid leukemia (AML) and multiple myeloma (MM) are two types of blood cancers with a high unmet therapeutic need. The knowledge that cells of our immune system can recognize and kill cancer cells has laid the foundation for immune effector cell (IEC) therapy. This involves the infusion of immune cells that are "armored" outside the body with a T-cell receptor (TCR) or a chimeric antigen receptor (CAR). Such TCR- or CAR-loaded immune cells can execute a targeted attack against cancer cells. The aim of the present project is to improve the therapeutic efficacy of IEC therapy for AML and MM, while reducing the risk of side effects and costs of treatment. More specifically, immune cells will be weaponed with AML-directed TCRs or MM-directed CARs via a technique called electroporation. This involves the application of an electrical pulse to the cells, making temporary holes in their surface and enabling their loading with the TCR or CAR. When compared to the current IEC therapies, this novel procedure will allow for the generation of IECs with reduced costs, improved safety profile and enhanced anti-tumor activity. It is therefore expected that this research project will make an important contribution to the development of the next-generation IEC products for AML and MM.

Researcher(s)

• Promoter: Anguille Sébastien
• Promoter: Van Tendeloo Vigor
• Co-promoter: Berneman Zwi
• Fellow: Anguille Sébastien

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Acute myeloid leukemia (AML) is an agressive type of blood cancer that still carries a dreadful prognosis. Even with treatment, only one out of 4 patients with AML will be alive 5 years after the diagnosis. This explains the urgent need for novel therapies. It is well known that our own immune system can fight cancer, laying the foundation for the development of immune-based therapies for cancer. One type of immunotherapy that has attracted much recent interest is T-cell therapy. Such therapy is based on the intrinsic ability of T cells - an important part of our immune system - to recognize and kill cancer cells. They do so via T-cell receptors, which are expressed on their cell surface. These T-cell receptors recognize certain substances, called antigens, that are presented by the cancer cells. In the context of AML, one antigen that serves as an attractive T-cell target is the Wilms' tumor protein 1 (WT1). In this research project, we will try to "weaponise" T cells to attack leukemia cells by genetically enforcing the expression of T-cell receptors against WT1. The ultimate goal is to exploit these anti-leukemia "killer" T cells for therapeutic purposes and to fulfill the unmet therapeutic need in AML.

Researcher(s)

• Promoter: Anguille Sébastien

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

This research aims to develop new strategies to enhance the immune stimulating and antitumor activity of dendritic cell therapy in acute myeloid leukemia.

Researcher(s)

• Promoter: Anguille Sébastien

Research team(s)

Project type(s)

• Research Project

Abstract

The specific aim of this project is to outline and experimentally validate new approaches to leverage the immunotherapeutic potential of DCs in the setting of malignancy. Both direct and indirect strategies to enhance the immunostimulatory capacity of DCs will be investigated.

Researcher(s)

• Promoter: Berneman Zwi
• Fellow: Anguille Sébastien

Research team(s)

Project type(s)

• Research Project

Abstract

The subject of this PhD project has to be situated within the framework of existing research activities at the Laboratory of Experimental Haematology focusing on the effects of innate immune signals on leukaemic tumor cells. This project aims to augment the immunogenicity of leukaemic cells through Toll-like receptor stimulation in order to (1) modulate the maturation stage of dendritic cells and to (2) subsequently potentiate a powerful acute myeloid leukemia-specific T-cell response.

Researcher(s)

• Promoter: Berneman Zwi
• Fellow: Anguille Sébastien

Research team(s)

Project type(s)

• Research Project