HA-1-targeted T-cell receptor T-cell therapy for recurrent leukemia after hematopoietic stem cell transplantation.

ABSTRACT: Relapse is the leading cause of death after allogeneic hematopoietic stem cell transplantation (HCT) for leukemia. T cells engineered by gene transfer to express T cell receptors (TCR; TCR-T) specific for hematopoietic-restricted minor histocompatibility (H) antigens may provide a potent selective antileukemic effect post-HCT. We conducted a phase 1 clinical trial using a novel TCR-T product targeting the minor H antigen, HA-1, to treat or consolidate treatment of persistent or recurrent leukemia and myeloid neoplasms. The primary objective was to evaluate the feasibility and safety of administration of HA-1 TCR-T after HCT. CD8+ and CD4+ T cells expressing the HA-1 TCR and a CD8 coreceptor were successfully manufactured from HA-1-disparate HCT donors. One or more infusions of HA-1 TCR-T following lymphodepleting chemotherapy were administered to 9 HCT recipients who had developed disease recurrence after HCT. TCR-T cells expanded and persisted in vivo after adoptive transfer. No dose-limiting toxicities occurred. Although the study was not designed to assess efficacy, 4 patients achieved or maintained complete remissions following lymphodepletion and HA-1 TCR-T, with 1 patient still in remission at >2 years. Single-cell RNA sequencing of relapsing/progressive leukemia after TCR-T therapy identified upregulated molecules associated with T-cell dysfunction or cancer cell survival. HA-1 TCR-T therapy appears feasible and safe and shows preliminary signals of efficacy. This clinical trial was registered at ClinicalTrials.gov as #NCT03326921.

CBFB-MYH11 fusion neoantigen enables T cell recognition and killing of acute myeloid leukemia.

Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11-specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope-specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low-mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene-driven AML.

T-Cell Receptor-Based Immunotherapy for Hematologic Malignancies.

Adoptive immunotherapy with engineered T cells is at the forefront of cancer treatment. T cells can be engineered to express T-cell receptors (TCRs) specific for tumor-associated antigens (TAAs) derived from intracellular or cell surface proteins. T cells engineered with TCRs (TCR-T) allow for targeting diverse types of TAAs, including proteins overexpressed in malignant cells, those with lineage-restricted expression, cancer-testis antigens, and neoantigens created from abnormal, malignancy-restricted proteins. Minor histocompatibility antigens can also serve as TAAs for TCR-T to treat relapsed hematologic malignancies after allogeneic hematopoietic cell transplantation. Moreover, TCR constructs can be modified to improve safety and enhance function and persistence of TCR-T. Transgenic T-cell receptor therapies targeting 3 different TAAs are in early-phase clinical trials for treatment of hematologic malignancies. Preclinical studies of TCR-T specific for many other TAAs are underway and offer great promise as safe and effective therapies for a wide range of cancers.

Intracellular Nucleic Acid Sensing Triggers Necroptosis through Synergistic Type I IFN and TNF Signaling.

The sensing of viral nucleic acids within the cytosol is essential for the induction of innate immune responses following infection. However, this sensing occurs within cells that have already been infected. The death of infected cells can be beneficial to the host by eliminating the virus's replicative niche and facilitating the release of inflammatory mediators. In this study, we show that sensing of intracellular DNA or RNA by cGAS-STING or RIG-I-MAVS, respectively, leads to activation of RIPK3 and necroptosis in bone marrow-derived macrophages. Notably, this requires signaling through both type I IFN and TNF receptors, revealing synergy between these pathways to induce cell death. Furthermore, we show that hyperactivation of STING in mice leads to a shock-like phenotype, the mortality of which requires activation of the necroptotic pathway and IFN and TNF cosignaling, demonstrating that necroptosis is one outcome of STING signaling in vivo.

Kinase-independent feedback of the TAK1/TAB1 complex on BCL10 turnover and NF-κB activation.

Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.

Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors.

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications.