Research Diana Campillo Davó

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

Prospects for cancer patients are certainly ameliorated in the past decade, especially thanks to the advances in the field of immunotherapy. However, it is important to note that many challenges remain. More specific, the majority of patients in need of better treatment options does not respond to immune checkpoint blocking therapy while CAR T cell therapy has only limited efficacy against solid tumours and on top causes severe side effects. Also, many tumours produce immunosuppressive cytokines like TGFβ, hampering current immunotherapy approaches. To address the challenges, our lab has developed over the past five years a TGFβ-resistant, Interleukin-15 producing CAR NK cell therapy which targets the CD70 protein. CAR NK cells have the important advantage that – contrary to CAR T cells – they do not cause severe side effects. Moreover, they can be produced as off-the-shelf therapy and do not require the same cumbersome production process as CAR T cells. The CD70 protein is also a ideal target since it is highly expressed in both leukaemia and solid tumours. This project has two main objectives. First, we want to gather the necessary preclinical data showing that our CAR NK cells are also successful against acute myeloid leukaemia. Secondly, we want to scale-up the production process of our CAR NK cells from research-grade to GMP-grade as this is a requirement for entering the clinical trial stage.

Researcher(s)

• Promoter: Smits Evelien
• Co-promoter: Campillo Davó Diana
• Co-promoter: De Waele Jorrit
• Co-promoter: Lion Eva
• Co-promoter: Van Audenaerde Jonas

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Next Generation Sequencing (NGS) has emerged as a suitable tool to evaluate and characterize the T Cell Receptor (TCR) immune repertoire. This approach paves the way for the use of the TCR repertoire study as a novel complex biomarker to track the exposure of individuals to non-self antigens and to develop new therapeutic strategies by improving our comprehension of the adaptive immune response against infectious agents and cancer antigens. The collaboration between Italy's Istituto Romagnolo per lo Studio dei Tumori "Dino Amadori" (IRST) and Belgium's University of Antwerp involves both these fields and aims to identify and characterize specific human TCRs via NGS to develop therapeutic TCR-T cells, exploiting experimental protocols, in silico analysis, and in vitro assays for T cell stimulation with antigens' pools. The first objective is to study the T cell response to mRNA-based COVID vaccines in healthy subjects and lymphoma patients from Emilia Romagna region through RNA samples sequencing and subsequent analysis of TCR specificity against SARS-CoV-2 proteins. The correlation between TCR diversity and strength with the amount of neutralizing antibodies will also be examined in consideration of the predicted HLA context, leading to an actionable SARS-Cov2 bulk TCR-seq database. Genomic instability, which is a common feature in cancer cells, often leads to the generation of chromosomal rearrangements and aneuploidy. Many are the examples today of gene fusions that promote cell transformation in different oncological settings. Those events lead to the unique opportunity to generate neoantigens that could be presented to the immune system in an HLA-restricted manner. Therefore, the second objective is the identification and validation of neoantigens originated from genetic fusion events in cancer patients already available in IRST. Subsequently, experiments will be focused on the identification of antigen-specific T cells and TCRs against neoantigens originating from selected genetic fusion events. To these ends, computational protein reconstruction and prioritization from the already available fusion genes database will be performed to retrieve fusion transcripts in hematological tumors. These peptides will be later synthesized and pooled to stimulate T cells, with subsequent expansion and characterization of the expressed receptors. Before that, a proof-of-concept T cell expansion and following TCR-identification experiments with model protein(s) for a known neoantigen will be set up.

Researcher(s)

• Promoter: Lion Eva
• Co-promoter: Campillo Davó Diana
• Co-promoter: Meysman Pieter

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Recently, interest has grown in using self-amplifying RNA (saRNA) in vaccines for infectious diseases and cancer. SaRNA is a type of messenger RNA (mRNA) that contains the non-structural proteins of an alphavirus replicase complex that amplifies the original strand of RNA and allows the expression of proteins of interest in the host cell without risk of infection. Compared to conventional mRNA, saRNA-mediated expression of proteins of interest may last for 28 days, while lacks the risks of genomic integration or cell transformation of integrative technologies such as viral vectors, transposons, and CRISPR-based knock-in. Moreover, saRNA vaccines under clinical investigation show that saRNA is safe and elicits robust immune responses. However, this technology has not been explored yet to genetically engineer effector immune cells, such as T cells, ex vivo. Therefore, the START project aims to investigate and optimize saRNA transfection as an innovative and potent technology for genetically engineering T cells with chimeric antigen receptors (CARs) and T-cell receptors (TCR) against different hematological and solid cancer antigens, with a thorough evaluation of antitumor activity, T cell fitness and potential transcriptomic and cell metabolic changes that could be related to saRNA replication activity. The START project will provide the basis for the future generation of non-integrative and long-lasting CAR-T and TCR-T cell therapies for hematological and solid malignancies.

Researcher(s)

• Promoter: Lion Eva
• Fellow: Campillo Davó Diana

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Recently, interest has grown in using RNA in vaccines for infectious diseases and cancer. In this project, we will investigate a new type of messenger RNA (mRNA) for genetic engineering of T cells with chimeric antigen receptors (CARs) and T cell receptors (TCRs) against hematological and solid cancers. This novel mRNA allows prolonged expression of the protein of interest compared to conventional mRNA without the risks of insertional mutagenesis present in integrating engineering technologies such as viral transduction. We will conduct an in-depth analysis of T cell fitness after RNA engineering and thoroughly evaluate the antitumor activity of the RNA-engineered CAR-T and TCR-T cells. This project will provide the basis for the future generation of safer and more durable cellular therapies for hematological and solid cancers.

Researcher(s)

• Promoter: Campillo Davó Diana

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Recently, there has been growing interest in the use of self-amplifying mRNA (saRNA) in therapeutic vaccines for infectious diseases and cancer. SaRNA is a type of messenger RNA (mRNA) that contains the non-structural proteins (nsP1-4) of an alphavirus replicase that copies the original strand of mRNA upon delivery into the cell. The nsP1-4 replicon is followed by a subgenomic promoter and the sequence of a gene of interest, allowing the expression of proteins of interest in the host cell. The self-replicating property means that proteins of interest encoded in the transfected saRNA will be expressed for a longer period of time compared to conventional mRNA. However, since there is no integration into the genome of the host cell, insertional mutagenesis is prevented. Thus, saRNA-based strategies combine the best of stable viral- or non-viral-based and transient mRNA-based engineering strategies. SaRNA is usually delivered in vivo as "naked" saRNA with or without intradermal electroporation or formulated into nanoparticle vaccines, with which expression of the protein of interest may last for 28 days. However, the exploitation of this technology for ex vivo modification of T cells in a therapeutic product has never been explored thus far. The primary objective of the Replic-ON project is to explore saRNA transfection as an innovative technology for genetically engineering immune cells in the context of the development of cell-based therapies. If successful, this project will provide groundbreaking data for the further development of ex vivo saRNA transfection technology as an amenable approach for T-cell genetic engineering in larger fundamental research project applications. We expect that this project will be the cornerstone for the much-needed development of more efficient and long-lasting non-integrating cellular immunotherapies while straddling the boundary between short-lived conventional mRNA technologies and integrating technologies such as viral transduction. Finally, this pioneering research would consolidate our leadership on ex vivo saRNA-based cellular therapies within the research community.

Researcher(s)

• Promoter: Campillo Davó Diana

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

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: Campillo Davó Diana

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Human blood contains several immune-competent cells including cells of the innate and adaptive immune system. Over the past years, the phenotypic and functional boundaries distinguishing the main cell subsets of the immune system have become increasingly blurred. While it has been already established that T cells may share some phenotypic and functional features of natural killer (NK) cells, more recent evidence points to the existence of such overlap between NK cells and dendritic cells (DCs). Both NK cells bearing DC markers and antigen-presenting capacity and DCs expressing NK-related molecules and having cytotoxic functions have been described. In view of this, CD56, a prototypic marker of NK cells, was found to be expressed on DC derived from monocytes exposed to interleukin-15. We demonstrated that these IL15-DC were endowed with superior stimulatory and unique cytotoxic properties (killer DC). The aim of this project is to identify and characterize in detail the in vivo counterpart of these CD56+ killer DCs in human blood. Particular emphasis will be given to the reciprocal interactions of myeloid CD56+ DC-like cells with CD56+ innate lymphocytes (NK and NKT cells, γδ T cells) in the presence or absence of immunomodulatory molecules. Next, the capacity of CD56+ blood DCs to stimulate both innate and adaptive cell responses will be analyzed in a human acute myeloid leukemia (AML) model as a first step towards design of next-generation therapeutic AML vaccines.

Researcher(s)

• Promoter: Van Tendeloo Vigor
• Co-promoter: Lion Eva
• Fellow: Campillo Davó Diana

Research team(s)

Project type(s)

• Research Project