Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological diseases

The research within our LUMC departments is conducted within departmental research programmes. The research programme below is embedded within the department of Hematology.

Aim and focus

The natural behavior of hematological diseases and their responses to targeted therapeutic interventions are governed by recurrent and individual pathogenic drivers. In addition, inherited and adaptive immune mechanisms of the host, and of the stem cell donor in the case of allogeneic stem cell transplantation (SCT), play decisive roles for success and outcome of therapy.

The research program of the Department of Hematology pursues the vision to

  • Exploit advanced molecular profiling for mechanistic understanding of molecular and immunological pathogenic factors to individually optimize therapeutic interventions
  • Unravel mechanisms of hematopoiesis- and malignancy-directed immune reactivity
  • Develop novel diagnostic and bench-to-bedside therapeutic strategies based on this new knowledge.

Using clinical observations and bio materials as starting points, research questions are being addressed in the Laboratory of Experimental Hematology. Basic research is then combined with translational research allowing the identification of crucial targets and mediators of immunopathogenesis, of successful cellular immunotherapy, and of the pathogenesis of detrimental toxicity of immunotherapy.

Common basis to realize these visions is a detailed molecular and cellular characterization of hematological tumors and of the host’s immune system.

To this end, research projects include:

  • Implementation of RNAseq and development of the HAMLET bioinformatics pipeline as a comprehensive diagnostic tool for AML.
  • Development and implementation of an advanced targeted sequencing panel on formalin-fixed tissue for molecular characterization of lymphoid malignancies in a consortium with the nonacademic hospitals in the region.
  • Unbiased molecular characterization of the repertoire of B-cell and T-cell receptors (BCR, TCR) in health, neoplastic, and autoimmune conditions by massive parallel, single-molecule next-generation sequencing. This project actively contributes to research projects of the Departments of Rheumatology, Dermatology, and Human Genetics.

The largest and longest-standing research efforts of the Department of Hematology are coupled to the TRF of allogeneic SCT. Allogeneic SCT with its elements of cellular immunotherapy and humoral immunotherapy has an essential role in the treatment of many malignant and non-malignant hematological disorders. Despite major improvements in recent years allowing application of this treatment in patients up to 75 years of age, improvement of efficacy and reduction of toxicity of allogeneic SCT and are still required.

The concept of cellular immunotherapy in the context of allogeneic SCT has been accepted as one of the most potent curative treatments of many hematological cancers. As the main therapeutic advantage of allogeneic SCT, adoptive cellular immunotherapy can lead to long-term control of hematological malignancies by specific immune surveillance in vivo.

Furthermore, strategies developed in the context of allogeneic SCT can lead to new therapeutic applications of autologous cellular immunotherapy, and may lead to new insights into the immunobiology and treatments of hematological autoimmune disorders like aplastic anemia. 

To offer access to the curative treatment option of allogeneic SCT to elderly patients and patients with comorbidities, investigator-initiated trials of the Department of Hematology address:

  • The tolerability and efficacy of less intensive induction regimens for AML
  • The amalgamation of AML consolidation therapy and reduced-intensity conditioning for allogeneic SCT into a single treatment regimen

For immune interventions, either post-transplant or as an alternative treatment strategy, the Department of Hematology studies the immunobiology and concepts of cellular immunotherapy from bedside to bench and from bench to bedside.

To achieve these goals, research projects include:

  • Development of new methods of conditioning, graft manipulation and post-transplant interventions to improve cellular immunotherapy in the context of allogeneic SCT. In collaboration with national partners, new transplantation platforms are being developed to allow controlled post-transplant immune interventions.
  • Characterization of the T-cell repertoire, T-cell biology and immune reconstitution after allogeneic SCT and cellular immunotherapy. By combining detailed clinical characteristics with in-depth laboratory analysis of immune responses in patients, new immunological phenomena are being discovered.
  • The characterization of target structures and quality and quantity of T-cell responses against these target structures involved in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) responses. This analysis allows to potentially separate the beneficial GVL responses from the detrimental GVHD reactivity.
  • The characterization of mechanisms leading to desired resistance of non- hematopoietic tissues and non-desired resistance of leukemic (stem) cells for cellular immunotherapeutic interventions. The characterization of in vivo checkpoints and resistance mechanisms will lead to new interventions promoting the beneficial immune response after allogeneic SCT.
  • Structural analysis of T-cell recognition and T-cell repertoire to allow definition of specific on-target and off-target specificity of T-cell receptors. This basic research program we lead to better understanding of the delicate balance between beneficial on target specificity, and undesired cross-reactivity with special focus on the application of T-cell receptor gene transfer.
  • The development of in vitro T-cell responses against defined antigens including pathogens, minor histocompatibility antigens and tumor-associated antigens for specific adoptive T-cell therapy. This translational research program concerns development of isolation under good manufacturing practice (GMP) conditions of antigen-specific T cells by magnetic bead isolation following specific activation of desired T cells, or following binding to peptide/MHC complexes.
  • The development of identification and isolation of high affinity T-cell receptors from T-cell responses against lineage-specific antigens or tumor-specific neo antigens for the treatment of hematological malignancies in the autologous setting. These TCRs are searched for in the context of the autologous T-cell repertoire, as well as the allogeneic HLA T-cell repertoire derived from healthy donors.
  • Translational clinical research to allow clinical application of in vitro selected donor derived pathogen-specific T cells for the control of viral reactivations after transplantation and in immune compromised hosts. These T-cell responses include complex products directed against multiple antigens from various viruses like CMV, EBV, and ADV.
  • Translational clinical research to allow clinical application of in vitro selected or cultured donor T cells specific for minor histocompatibility antigens or tissue restricted alloantigens and T-cell receptor-transduced virus-specific T cells for the treatment of hematological disorders in the context of allogeneic SCT.
  • Translational research to allow clinical application of genetically modified T cells by T-cell receptors specific for lineage-associated antigens or neoantigens for the treatment of hematological disorders.
  • The development of new strategies of combined cellular and humoral immunotherapy including checkpoint inhibition for the treatment of hematological malignancies.

Disease-specific research projects addressing molecular immunopathogenesis and immunotherapy of hematologic diseases include:

  • Dissection of the functional microarchitecture of follicular lymphoma with the goal of establishing the hierarchy and functional interaction of immunological drivers and molecular oncogenesis. Given its chronic protracted course and continuous accumulation of somatic hypermutations, follicular lymphoma is paradigmatic tumor to study the mechanisms of secondary oncogenic drivers.
  • Defining the role of stochastically occurring, autonomous BCR signaling in the pathogenesis of CLL and aggressive B-cell lymphomas with focus on the acquisition of this signal dependent the cell of origin and its role in progression from clonal expansion to frank malignancy in the MBL – CLL paradigm.
  • Basic mechanisms and immunological efficacy of active immunization with tumor-individual B-cell receptor peptide for the treatment of indolent B-cell malignancies.
  • The development of LUMC as expertise center for aplastic anemia, the development of the national registry for diagnostics, treatment and monitoring of patients with aplastic anemia.
  • Identification of the immunobiology of aplastic anemia, and optimization of humoral and pharmacological immunosuppressive therapy for acquired aplastic anemia and the potential of stem cell-stimulating combination therapy.

Position in international context

Our combined bedside-to-bench and bench-to-bedside research program covering clinical research, translational research and basic research holds an internationally highly recognized position.

With the excellent infrastructure of the Leiden Genome Technology Center (LGTC) and of the Sequence Analysis Support Core (SASC), the Department has gained a technological advantage for comprehensive analysis of immune receptor repertoires in health and disease. The accredited infrastructure of GenomeScan BV is an essential element to being a forerunner in bringing whole transcriptome sequencing to routine diagnostic application.

The continuous expansion of the departmental cellular biobank to currently almost 500,000 cryopreserved viable cell samples is a valuable and exceptional resource to study molecular and immunopathogenesis of hematologic malignancies on primary human cells. In close collaboration with H. Jumaa, Institute of Immunology at Ulm University as a basic research partner, the Department of Hematology has an internationally unique research line to unravel autonomous BCR signaling as a novel paradigm in lymphomagenesis. 

In unique combination with detailed databases containing clinical characteristics, the biobank is essential for in-depth analysis of immune responses of patients following allogeneic SCT and cellular immunotherapy.

The close collaboration with the Department of Clinical Pharmacology and the availability of a well-equipped GMP facility facilitates the development and conduct of investigator-initiated trials that investigate safety and efficacy of investigator-created novel clinical cellular products. This infrastructure and expertise provides an attractive platform for collaborations with international groups and companies. Only a limited number of other centers in the USA, Great Britain and Germany have similar extended programs allowing full coverage of basic, translational and clinical research on immunology and immunotherapy of hematological diseases. Collaborations with national and international partners allow optimal use of materials collected and developed in the context of our immune intervention program.

The national leadership gained by implementing and running the Dutch registry for aplastic anemia with endorsement and support from the Netherlands Association for Hematology (NVvH) enables the Department of Hematology to perform internationally recognized population-based studies on efficacy and long-term outcome of established and novel therapies. This position also makes the Department of Hematology a leading player with respect to multinational trials on aplastic anemia at the European level within the EBMT framework.

With respect to valorization of research results, a structural collaboration with Bellicum Pharmaceuticals towards the preclinical and clinical development of TCR-transgenic T cell therapy represents an outstanding example with a financial volume of international rank.

Content / highlights / achievements

Clinical innovation and Value-Based Health Care:

  • A low risk procedure of allogeneic SCT including in vitro and in vivo T cell depletion using alemtuzumab followed by cellular immunotherapy strategies has been developed allowing sibling and unrelated transplants in patients up to 75 years of age. The absence of severe GVHD and the absence of the necessity to use long-term immunosuppressive agents following transplantation provide a platform for the application of innovative cellular therapeutic interventions post-transplant.
  • The introduction of intensive therapeutic approaches for primary CNS lymphoma, to the Netherlands, including high-dose chemotherapy with autologous SCT for younger patients.
  • Creation of the national registry concerning new therapeutic interventions and monitoring to improve treatment of patients with severe aplastic anemia. The national collaboration has been established to optimize treatment and monitoring of patients with a plastic anemia.
  • A clinical protocol for the treatment for CMV disease after allogeneic SCT with in vitro cultured CMV-specific T cells has been installed. The successful development of this method has led to hospital exemption approval for the production and application of CMV-specific T cells for the treatment of resistant CMV reactivity following allogeneic SCT and solid organ transplantation.

Diagnostic innovation:

  • Development of the HAMLET pipeline for RNAseq data as comprehensive genetic diagnostics for AML to replace conventional batteries of separate tests.

Basic discoveries:

  • Discovery of many new minor histocompatibility antigens resulted in the highest number of minor histocompatibility antigens worldwide. A new high throughput discovery method has been developed allowing to further characterize the panel of minor histocompatibility antigens that can be used to stratify and select optimal donors for allogeneic SCT.
  • Discovery of the influence of the HLA type on the normal B-cell repertoire
  • Description of the IgE repertoire in comparison to IgM/IgG repertoires in healthy individuals
  • Discovery of autonomous BCR signaling as potential pathogenic driver in aggressive B-cell malignancies
  • Characterization of the role of Aspergillus-specific T cells  in invasive aspergillosis (in collaboration with the Department of Infectious Diseases)

Translational developments:  

  • Characterization of the allo-immune repertoire and specificity in HLA matched and HLA mis-matched transplantation. Correlation of the characteristics of the allogeneic immune response with clinical outcome provides better insight into the biology of allogeneic SCT, and provides new tools for specific targeting of hematological malignancies.
  • We developed a high throughput method to isolate and characterize high avidity tumor antigen or lineage specific T cells from the allogeneic HLA repertoire of patients and healthy individuals. This has led to the identification of a variety of T-cell responses specific for B cell lineage associated antigens that can be targeted by TCR gene transfer for the treatment of B-cell malignancies and multiple myeloma. Thus far, patents have been filed, and a licensing contract has been signed with a pharmaceutical company for the clinical development of T-cell receptor gene transfer targeting PRAME and BOB-1.
  • We developed a method to define, isolate and characterize high avidity T cells from healthy individuals directed against leukemia specific neo antigens. By combining gene expression profiles with peptidome analysis of hematological malignancies, specific target neoantigens has been characterized, T-cell responses have been generated and TCRs that can be used for T-cell receptor gene transfer have been identified. A patent application is being filed.

Investigator-initiated trials based on own bench-to-bedside developments:

  • The development of randomized studies of adoptive immunotherapy post allogeneic SCT using purified CD4-positive T cells for prevention and treatment of complications and recurrence of the disease after transplantation allows the specific characterization of the role of CD4 T cells in the context of transplantation and cellular therapy, the relevance of HLA class II target antigens in allogeneic SCT and cellular therapy, and the development of new therapeutic targets to use antigen-specific CD4 T cells to control hematological disorders and pathogens in the context of transplantation.
  • We developed a clinical protocol for the treatment of viral reactivations following allogeneic SCT by multi-antigen specific T cells. A clinical trial has been executed using application of CD8 T cells simultaneous isolated using streptamers specific for multiple antigens from various viruses and tumor associated antigens for application early after T cell depleted transplantation.
  • Development of redirection of viral specific T cells with antigen specific T cell receptor using T cell receptor gene transfer: from basic research to clinical application: an investigator initiated clinical trial is being executed using the HA-1 specific TCR transduced into EBV or CMV specific T cells isolated and activated using peptide/MHC specific streptamers for the treatment of high-risk leukemia following allogeneic SCT.

Future themes

  • Development of disease-directed bioinformatics pipelines (HALLET, HuMDySET) and implementation of RNAseq for routine diagnostics of ALL and MDS (in collaboration with LGTC/SASC).
  • Development and implementation of advanced targeted sequencing panels for B- and T-cell malignancies throughout the Leiden-Den Haag region (in collaboration with all regional nonacademic hospitals).
  • Investigation of the relative role and hierarchical position of autonomous BCR signaling in the lymphomagenesis of CLL and aggressive B-cell lymphomas, including translation of this knowledge towards BCR signaling blockade as a novel therapeutic modality in selected aggressive (cutaneous) B-cell lymphomas.
  • Deciphering the hierarchy and functional interaction between immunologic and genetic mechanisms that govern the biological behavior of follicular lymphoma as a paradigmatic B-cell malignancy.
  • Large scale identification and molecular characterization of minor histocompatibility antigens for the selection and stratification of donors for allogeneic SCT and cellular immunotherapy. Approximately 200 to 500 new minor histocompatibility antigens will be characterized, a database using these antigens will be generated and provided to the community to characterize the role of minor antigens in allogeneic SCT and cell therapy using large clinical databases.
  • Biochemical characterization of the peptidome presented in HLA molecules from normal cell populations for the identification of permissive and non-permissive donors for allogeneic SCT and targeting of beneficial immune responses directed against peptides presented in allogeneic HLA molecules present on cells of hematopoietic origin. This program will be performed in collaboration with the University of Essen, Germany.
  • Identification of lineage specific or tumor specific neo antigens in the peptidome presented in HLA molecules from malignant cell populations to develop specific targeting of hematological tumors by T cell receptor gene transfer. Low-frequency high avidity T cells will be isolated from healthy donors and patients, analyzed and characterized, the relevant TCR will be identified and patent applications will be filed to allow both academic and industrial development of these TCR.
  • T cell receptor gene transfer as a method to redirect T cells specific for lineage specific antigens, tumor associated antigens and tumor-specific neo antigens for the treatment of hematological malignancies both in the context of allogeneic SCT and without SCT. This will be developed both as investigator initiated trials, as well as collaborative trials with industry
  • Characterization of the high avidity allogeneic T-cell repertoire capable of recognizing target antigens of interest to develop new cellular therapeutic strategies. There will be special focus on the characterization and isolation of multi lineage hematopoiesis restricted antigen specific CD4 T cells capable of targeting HLA class II expressing hematological malignancies.
  • A national clinical transplantation platform will be developed using T-cell depleted allogeneic transplantation for the application of post-transplant cellular interventions.
  • Development of vaccination protocols for in vivo activation of tumor-reactive T cell responses. A unique program is the reinvigoration of BCR neoantigen-specific anti-lymphoma vaccination based on synthetic peptides.
  • The characterization and exploration of new strategies to combine humoral and cellular immune therapy of hematological disorders. This also includes the combination of cell therapy with checkpoint inhibitory antibodies.
  • Development of new strategies to treat refractory hematological malignancies with autologous T cells modified using TCR gene transfer directed against the lineage specific antigens, tumor associated antigens or tumor associated neoantigens. These strategies include TCR specific for PRAME, Bob1, and the newly discovered AML specific neo antigen.
  • Further development of the LUMC as expertise center for aplastic anemia, development of new therapeutic interventions for aplastic anemia, and characterization of the immune biology of the disease, and the mechanism of action of ATG.
  • Characterization of the human IgE repertoire in allergic conditions (in collaboration with the Department of Dermatology).

Cohesion within LUMC

The research program as a whole is embedded in the Medical Research Profile Cancer Pathogenesis and Therapy (CPT).

The diagnostic RNAseq and targeted lymphoma panel efforts strengthen the CPT Focus Area on Cancer Genetics and Epigenetics. For application of these results to therapeutic choices for individual patients, the Department of Hematology actively participates in the multidisciplinary Molecular Tumor Board and can take advantage of the Leiden Network on Personalized Therapeutics (LNPT). These activities fall under the CPT Focus Area on Molecular Therapeutics. The CPT Focus Area on Cancer Immunotherapy receives the strongest input from the Department of Hematology. The HLA ligandome characterization projects that are conducted in collaboration with the Technology Focus Area (TFA) Center for Proteomics and Metabolomics (CPM) form a strong aspect of the proteomic studies within CPT. The knowledge created on initiation, regulation, and effector mechanisms of antigen-specific T-cell responses that arise in the allogeneic SCT setting complement and broaden the projects investigating anti-tumor T-cell immunity within CPT. In turn, the Department of Hematology will benefit from the developments of highly immunogenic synthetic peptide vaccines and from the expertise gained within CPT with checkpoint inhibition and resulting neoepitope-specific  T-cell responses.

The translational research efforts for bench-to bedside development of novel therapies, in particular cellular immunotherapy products, are facilitated by a network that has been built within the LUMC including the Departments of Hematology, Clinical Pharmacology and Toxicology (GMP facility), Immunohematology and Blood Transfusion, including the center for Stem Cell Therapy, the TFA CPM, and the LUMC immunomonitoring platform.

A collaborative project within CPT is being conducted together with the Department of Dermatology on the pathogenesis and treatment of cutaneous lymphomas where the LUMC represents the National Expertise Center.

Within the LUMC Oncology Center, the Department of Hematology organizes and runs the tumor theme working group on hematologic malignancies together with the Department of Radiation Therapy and the diagnostic disciplines of the Departments of Pathology, Clinical Genetics, Radiology, and Nuclear Medicine. With respect to focusing on TRF functions, inclusion rates in clinical trials, tumor biobanking, own investigator-initiated trials, and intrinsically linked laboratory-based research, hemato-oncology fulfils all essential criteria for a successful tumor theme group within the LUMC Oncology Center.

The expertise of the Department of Hematology with respect to immune repertoire, HLA ligandome, and specific T-cell responses contributes to the Medical Research Profile Immunity, Infection, and Tolerance (IIT). Specific joint projects address the immunopathogenesis of myasthenia gravis (in collaboration with the Department of Human Genetics) and of rheumatoid arthritis (in collaboration with the Department of Rheumatology).