Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14+ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34+ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

Transcriptional behavior of the HIV-1 promoter in context of the BACH2 prominent proviral integration gene.

Chronic infection with human immunodeficiency virus (HIV)-1 is characterized by accumulation of proviral sequences in the genome of target cells. Integration of viral DNA in patients on long-term antiretroviral therapy selectively persists at preferential loci, suggesting site-specific crosstalk of viral sequences and human genes. This crosstalk likely contributes to chronic HIV disease through modulation of host immune pathways and emergence of clonal infected cell populations. To systematically interrogate such effects, we undertook genome engineering to generate Jurkat cell models that replicate integration of HIV-1 long terminal repeat (LTR) sequences at the BTB and CNC Homolog 2 (BACH2) integration locus. This locus is a prominent HIV-1 integration gene in chronic infection, found in 30 % of long-term treated patients with mapped proviral integrations. Using five clonal models carrying an LTR-driven reporter at different BACH2 intergenic regions, we here show that LTR transcriptional activity is repressed in BACH2 regions associated with proviral-DNA integrations in vivo but not in a control region. Our data indicates that this repression is in part epigenetically regulated, particularly through DNA methylation. Importantly, we demonstrate that transcriptional activity of the LTR is independent of BACH2 gene transcription and vice versa in our models. This suggests no transcriptional interference of endogenous and HIV-1 promoters. Taken together, our study provides first insights into how activity of HIV-1 LTR sequences is regulated at the BACH2 locus as prominent example for a recurrently-detected integration gene in chronic infection. Given the importance of integration-site dependent virus/host crosstalk for chronic HIV disease, our findings for the BACH2 locus have potential implications for future therapeutic strategies.

PD-1 Blockade Aggravates Epstein-Barr Virus+ Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4+ T Cell Dysregulations.

Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV+ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81+ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34+ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8+ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67+CD30+CD20+EBER+PD-L1+ PTLD with central nervous system (CNS) involvement, mirroring EBV+ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4+ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3+ regulatory CD4+ and CD8+ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8+ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV+ PTLD.

Monocytes reprogrammed with lentiviral vectors co-expressing GM-CSF, IFN-α2 and antigens for personalized immune therapy of acute leukemia pre- or post-stem cell transplantation.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and overall survival remains poor. Chemotherapy is the standard of care for intensive induction therapy. Patients who achieve a complete remission require post-remission therapies to prevent relapse. There is no standard of care for patients with minimal residual disease (MRD), and stem cell transplantation is a salvage therapy. Considering the AML genetic heterogeneity and the leukemia immune-suppressive properties, novel cellular immune therapies to effectively harness immunological responses to prevent relapse are needed. We developed a novel modality of immune therapy consisting of monocytes reprogrammed with lentiviral vectors expressing GM-CSF, IFN-α and antigens. Preclinical studies in humanized mice showed that the reprogrammed monocytes self-differentiated into highly viable induced dendritic cells (iDCs) in vivo which migrated effectively to lymph nodes, producing remarkable effects in the de novo regeneration of T and B cell responses. For the first-in-man clinical trial, the patient's monocytes will be transduced with an integrase-defective tricistronic lentiviral vector expressing GM-CSF, IFN-α and a truncated WT1 antigen. For transplanted patients, pre-clinical development of iDCs co-expressing cytomegalovirus antigens is ongoing. To simplify the product chain for a de-centralized supply model, we are currently exploring a closed automated system for a short two-day manufacturing of iDCs. A phase I clinical trial study is in preparation for immune therapy of AML patients with MRD. The proposed cell therapy can fill an important gap in the current and foreseeable future immunotherapies of AML.