Leukemia relapse remains the major cause of allogeneic hematopoietic stem cell transplantation (HCT) failure, and the prognosis for patients with post-HCT relapse is poor. There is compelling evidence that potent selective antileukemic effects can be delivered by donor T cells specific for particular minor histocompatibility (H) antigens. Thus, T-cell receptors (TCRs) isolated from minor H antigen-specific T cells represent an untapped resource for developing targeted T-cell immunotherapy to manage post-HCT leukemic relapse. Recognizing that several elements may be crucial to the efficacy and safety of engineered T-cell immunotherapy, we developed a therapeutic transgene with 4 components: (1) a TCR specific for the hematopoietic-restricted, leukemia-associated minor H antigen, HA-1; (2) a CD8 coreceptor to promote function of the class I-restricted TCR in CD4 T cells; (3) an inducible caspase 9 safety switch to enable elimination of the HA-1 TCR T cells in case of toxicity; and (4) a CD34-CD20 epitope to facilitate selection of the engineered cell product and tracking of transferred HA-1 TCR T cells. The T-cell product includes HA-1 TCR CD4 T cells to augment the persistence and function of the HA-1 TCR CD8 T cells and includes only memory T cells; naive T cells are excluded to limit the potential for alloreactivity mediated by native TCR coexpressed by HA-1 TCR T cells. We describe the development of this unique immunotherapy and demonstrate functional responses to primary leukemia by CD4 and CD8 T cells transduced with a lentiviral vector incorporating the HA-1 TCR transgene construct.

BackgroundRecent studies have demonstrated that several mineral products sold for medicinal purposes demonstrate antimicrobial activity, but little is known about the physicochemical properties involved in antibacterial activity.Methodology/Principal FindingsUsing in vitro mineral suspension testing, we have identified two natural mineral mixtures, arbitrarily designated BY07 and CB07, with antibacterial activity against a broad-spectrum of bacterial pathogens. Mineral-derived aqueous leachates also exhibited antibacterial activity, revealing that chemical, not physical, mineral characteristics were responsible for the observed activity. The chemical properties essential for bactericidal activity against Escherichia coli were probed by testing antibacterial activity in the presence of metal chelators, the hydroxyl radical scavenger, thiourea, and varying pH levels. Chelation of the BY07 minerals with EDTA or desferrioxamine eliminated or reduced BY07 toxicity, respectively, suggesting a role of an acid-soluble metal species, particularly Fe3+ or other sequestered metal cations, in mineral toxicity. This conclusion was supported by NMR relaxation data, which indicated that BY07 and CB07 leachates contained higher concentrations of chemically accessible metal ions than leachates from non-bactericidal mineral samples.Conclusions/SignificanceWe conclude that the acidic environment of the hydrated minerals significantly contributes to antibacterial activity by increasing the availability and toxicity of metal ions. These findings provide impetus for further investigation of the physiological effects of mineral products and their applications in complementary antibacterial therapies.

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.

BackgroundWe have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates.MethodsWe used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Baclight LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity.ResultsScanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates.ConclusionsThese data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.

Background: Donor T cells specific for minor histocompatibility (H) antigens can deliver potent, selective anti-leukemic effects after allogeneic HCT when the antigen is negligibly or not expressed by non-hematopoietic tissues, not present in the donor, and expressed by the recipient. We reported a new minor H antigen-directed T-cell therapy that can be deployed after HCT to manage persistent or recurrent measurable residual hematologic malignancies or overt relapse (Blood 2018;131(1):108). We developed a transgene with 4 components: 1) a high-affinity T-cell receptor (TCR) specific for the hematopoietic-restricted minor H antigen, HA-1 that is presented on HLA-A*02:01; 2) a CD8 co-receptor to enhance function of the class I-restricted TCR in CD4+ T cells so they promote cytotoxic CD8+ T cell function and survival; 3) an inducible caspase-9 safety switch, which can be triggered by the drug rimiducid in case of in vivo toxicity; and 4) a CD34-CD20 epitope to facilitate selection of the engineered product during manufacturing and track HA-1 TCR T cells in the recipient. The 21-day manufacturing process entails CD45RA+ naïve T cell depletion (minimizes the risk of GvHD), and subsequent CD4+ and CD8+ separation (provides a consistent 1:1 CD4:CD8 ratio). The separate cultures are transduced with the lentivirus construct iCasp9-HA1-TCR2-RQR-CD8, expanded, and selected using the CD34 marker to ensure removal of untransduced T cells. Study Design and Methods: The single-center phase I trial (NCT03326921) evaluates the feasibility and safety of infusion of HA-1 TCR T-cell immunotherapy. Primary end points are 1) Feasibility of manufacturing and administering HA-1 TCR CD8+ and CD4+ memory T cells and 2) Dose-limiting toxicity of HA-1 TCR T cells. Major inclusion criteria are: HLA-A*02:01-positive, HA-1-positive patients who underwent HCT for acute leukemia, myelodysplastic syndrome, BPDCN, CML, CMML or JMML from a HLA-A*-02:01+/HA-1-negative donor or HLA-A*02:01-negative haploidentical or mismatched donor (excluding umbilical cord). HA-1 genotype screening is performed on patient and donor blood, hair follicle or cheek swab samples shipped to Fred Hutchinson Cancer Research Center. To be eligible for treatment, patients must develop measurable residual disease or overt relapse after HCT but may receive other standard or investigational therapies prior to treatment with HA-1 TCR T-cell immunotherapy if clinically indicated. Some systemic immunosuppression may be continued, but prior grade IV acute GVHD and prior severe chronic GVHD are key exclusions. Two groups, <16 and ≥16 years, will be treated at dose levels ranging from 3 x 106 to30 x 106 cells/kg, in cohorts of 3-6 subjects, up to approximately 24 subjects in total. Fludarabine lymphodepletion will be used in most subjects, followed by a single T-cell infusion, with an option for a subsequent infusion(s) if the subject demonstrates an initial response without severe toxicity. Bone marrow aspirations are performed prior to T-cell infusion and at several time points following infusion. Recruitment and Patient Characteristics: To date, 3 subjects have been treated on the phase I clinical trial and received a total of 5 infusions (Table 1). HA-1 TCR T cell persistence in blood and bone marrow has been documented from >3 months to >13 months. Clear in vivo anti-leukemic activity was observed at the first dose level, including in a subject with aggressive, highly refractory T-ALL and early post-HCT relapse. Outlook: Minor H antigen-specific T-cell immunotherapy may offer effective management of post-HCT relapse while avoiding GvHD and other off-target effects. Due to population genetics of HA-1 and HLA-A*02:01, HA-1 TCR T-cell immunotherapy is applicable to 10-15% of HCT recipients with various hematological malignancies. The ongoing phase I trial is actively recruiting patients. Development of T-cell immunotherapy targeting other minor H antigen/HLA combinations is also underway to increase the broad applicability of minor H antigen-targeted T-cell immunotherapy. Disclosures Krakow: HighPass Bio: Research Funding. Cunningham:HighPass Bio: Research Funding. Vartanian:HighPass Bio: Research Funding. Bleakley:HighPass Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Although allogeneic hematopoietic stem cell transplantation (HCT) can cure patients with chemotherapy-refractory acute myeloid leukemia (AML), post-HCT relapse remains common and extremely challenging to resolve. T cell immunotherapy could augment HCT efficacy by inducing deeper remissions pre-HCT to prevent relapse, or to treat or prevent relapse post-HCT. However, development of T cell immunotherapy for AML has been limited by a paucity of antigens established to be safe and effective T cell targets. Recurrent AML-specific somatic variants, such as fusion genes and insertion-deletion mutations (indels), may produce novel amino acid sequences (neoepitopes) recognized by T cells. Because T cells recognize peptide epitopes presented in the context of HLA molecules, algorithms that predict peptide binding to HLA can be used to identify potential T cell epitopes from within a mutation-specific amino acid sequence. We propose to identify immunogenic AML-specific neoepitopes by merging next-generation sequencing (NGS) data of somatic leukemia-specific variants with established HLA-binding prediction algorithms, before performing a high-throughput immunogenicity screen. As proof-of-principle, we combined the output of 4 HLA-binding prediction algorithms to identify candidate neoepitopes with 12 prevalent HLA restrictions from 11 fusions and 8 indels identified by NGS from a cohort of w300 pediatric AML patients. We identified 96 peptide neoepitopes with 11 HLA-restrictions in 16 genomic anomalies. So far we have isolated CD8 + T cell clones specific for two HLA-A*02:01-restrictedneoepitopes, one from the inv(16) fusion, CBFB-MYH11, and one from the most prevalent NPM1 mutation. Titration of peptide concentration in a chromium-51 release assay revealed high functional avidity of the CBFB-MYH11 specific clone (Figure 1: CBFB-MYH11 peptide, solid line; irrelevant peptide, dashed line. Mean and SD of 3 experiments shown). Moreover, this clone specifically lysed primary inv(16)-positive AML blasts from an HLA-A*02:01 + patient, but did not significantly lyse blasts from an inv(16) + but HLA-A*0201patient (Figure 2; also shown are lysis of peptide-pulsed A2 + T2 cell line as controls). Targeted PCR sequencing of the CBFB-MYH11 fusion to determine the frequency of the fusion variant producing our candidate epitope in patients with inv(16) + AML is in progress. Ongoing studies will also determine the breadth and specificity of the CBFB-MYH11 specific clone's target recognition, and identify additional immunogenic neoepitopes using the methods described above. Our method detects naturally occurring AML-specific epitopes that may prove tractable T cell targets with which to develop novel immunotherapies for use before, during or after HCT.

149 Background: Allogeneic hematopoietic stem cell transplantation (HCT) often cures acute leukemia. However leukemic relapse remains a major cause of HCT failure, and patients with post-HCT relapse have a very poor prognosis. We are developing T cell immunotherapies targeting leukemia-associated minor histocompatibility (H) antigens to manage post-HCT relapse. Because the presentation of minor H antigens is HLA-restricted, a panel of hematopoietic-restricted, leukemia-associated minor H antigens is required to enable the development of broadly applicable minor H antigen targeted immunotherapy. We previously discovered a HLA- B*0801-restricted, leukemia-associated minor H antigen, HEATR1. We showed that HEATR1-specific T cells can specifically kill leukemic blasts and prevent engraftment in a murine model, implying that this minor H antigen is expressed in leukemic stem cells. We have now developed immunotherapy targeting HEATR1 using genetically modified T cells. Methods: We isolated a HEATR1-specific T cell clone from the peripheral blood of a normal donor and sequenced its T cell receptor (TCR). The TCR was codon-optimized and cysteine-modified to maximize expression in T cells and reduce the risk of mispairing with endogenous TCR chains, then cloned into a multicistronic lentiviral vector (LV). Primary CD8+T cells were transduced with the HEATR1 TCR LV and evaluated with a range of assays. Results: The HEATR1-specific T cell clone specifically killed B8+ HEATR1+ primary leukemia. Similarly, CD8+ HEATR1 TCR-transduced T cells killed target cells pulsed with HEATR1 peptide (ISKERAEAL), but not the allelic variant control peptide (ISKERAGAL), and specifically killed cell lines that endogenously present the HEATR1 minor H antigen. HEATR1-transduced T cells also secrete cytokines and proliferate in response to HEATR1+ but not HEATR1-cells. Conclusions: We have demonstrated that HEATR1-specific T cell clone and HEATR1 TCR-transduced T cells can kill HEATR1+ cells. If the safety and efficacy of HEATR1 TCR immunotherapy is confirmed in further preclinical studies, development of phase I clinical studies will be warranted and may ultimately provide a new option for management of relapse after HCT.