Multi-antigen intranasal vaccine protects against challenge with sarbecoviruses and prevents transmission in hamsters.

Immunization programs against SARS-CoV-2 with commercial intramuscular vaccines prevent disease but are less efficient in preventing infections. Mucosal vaccines can provide improved protection against transmission, ideally for different variants of concern (VOCs) and related sarbecoviruses. Here, we report a multi-antigen, intranasal vaccine, NanoSTING-SN (NanoSTING-Spike-Nucleocapsid), eliminates virus replication in both the lungs and the nostrils upon challenge with the pathogenic SARS-CoV-2 Delta VOC. We further demonstrate that NanoSTING-SN prevents transmission of the SARS-CoV-2 Omicron VOC (BA.5) to vaccine-naïve hamsters. To evaluate protection against other sarbecoviruses, we immunized mice with NanoSTING-SN. We showed that immunization affords protection against SARS-CoV, leading to protection from weight loss and 100% survival in mice. In non-human primates, animals immunized with NanoSTING-SN show durable serum IgG responses (6 months) and nasal wash IgA responses cross-reactive to SARS-CoV-2 (XBB1.5), SARS-CoV and MERS-CoV antigens. These observations have two implications: (1) mucosal multi-antigen vaccines present a pathway to reducing transmission of respiratory viruses, and (2) eliciting immunity against multiple antigens can be advantageous in engineering pan-sarbecovirus vaccines.

An intranasal nanoparticle STING agonist protects against respiratory viruses in animal models.

Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2'-3' cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.

Identification of a clinically efficacious CAR T cell subset in diffuse large B cell lymphoma by dynamic multidimensional single-cell profiling.

Chimeric antigen receptor (CAR) T cells used for the treatment of B cell malignancies can identify T cell subsets with superior clinical activity. Here, using infusion products of individuals with large B cell lymphoma, we integrated functional profiling using timelapse imaging microscopy in nanowell grids with subcellular profiling and single-cell RNA sequencing to identify a signature of multifunctional CD8+ T cells (CD8-fit T cells). CD8-fit T cells are capable of migration and serial killing and harbor balanced mitochondrial and lysosomal volumes. Using independent datasets, we validate that CD8-fit T cells (1) are present premanufacture and are associated with clinical responses in individuals treated with axicabtagene ciloleucel, (2) longitudinally persist in individuals after treatment with CAR T cells and (3) are tumor migrating cytolytic cells capable of intratumoral expansion in solid tumors. Our results demonstrate the power of multimodal integration of single-cell functional assessments for the discovery and application of CD8-fit T cells as a T cell subset with optimal fitness in cell therapy.

Sleeping beauty generated CD19 CAR T-Cell therapy for advanced B-Cell hematological malignancies.

Chimeric antigen receptor (CAR) T-cell therapy has emerged recently as a standard of care treatment for patients with relapsed or refractory acute lymphoblastic leukemia (ALL) and several subtypes of B-cell non-Hodgkin lymphoma (NHL). However, its use remains limited to highly specialized centers, given the complexity of its administration and its associated toxicities. We previously reported our experience in using a novel Sleeping Beauty (SB) CD19-specific CAR T-cell therapy in the peri-transplant setting, where it exhibited an excellent safety profile with encouraging survival outcomes. We have since modified the SB CD19 CAR construct to improve its efficacy and shorten its manufacturing time. We report here the phase 1 clinical trial safety results. Fourteen heavily treated patients with relapsed/refractory ALL and NHL were infused. Overall, no serious adverse events were directly attributed to the study treatment. Three patients developed grades 1-2 cytokine release syndrome and none of the study patients experienced neurotoxicity. All dose levels were well tolerated and no dose-limiting toxicities were reported. For efficacy, 3 of 8 (38%) patients with ALL achieved CR/CRi (complete remission with incomplete count recovery) and 1 (13%) patient had sustained molecular disease positivity. Of the 4 patients with DLBCL, 2 (50%) achieved CR. The SB-based CAR constructs allow manufacturing of targeted CAR T-cell therapies that are safe, cost-effective and with encouraging antitumor activity.

Cytotoxic T lymphocytes targeting a conserved SARS-CoV-2 spike epitope are efficient serial killers.

Understanding the cellular immune response to infections, cancers and vaccines lags behind the investigation of humoral responses. While neutralizing antibody responses wane over time, the ability of T cells to recognize viruses including SARS-CoV-2 is instrumental to providing long-term immunity. Although T-cell receptor (TCR) repertoire screening can provide insights into the skewing of a T-cell response elicited upon vaccination or infection, they unfortunately provide no assessment into the functional capacity of T cells or their ability to eliminate virally infected targets. We have used time-lapse imaging microscopy in nanowell grids (TIMING) to integrate the migration of individual T cells with analysis of effector functions including cytokine secretion and cytotoxicity. Machine learning is then applied to study thousands of videos of dynamic interactions as T cells with specificity for SARS-CoV-2 eliminate targets bearing spike protein as a surrogate for viral infection. Our data provide the first direct evidence that cytotoxic T lymphocytes from a convalescent patient targeting an epitope conserved across all known variants of concern (VoC) are serial killers capable of eliminating multiple infected targets. These data have implications for development of vaccines to provide broad and sustained cellular immunity and for the recovery and monitoring of individuals who have been exposed to SARS-CoV-2. MULTIDISCIPLINARY ABSTRACT: We present an imaging platform that uses artificial intelligence (AI) to track thousands of individual cell-cell interactions within nanowell arrays. We apply this platform to quantify how the T cell component of adaptive immunity responds to infections. Our results show that T cells specific for a conserved epitope within the SARS-CoV-2 spike protein are serial killers that can rapidly eliminate virally infected targets. The ability to map the functional capacity of T cells and their ability to kill infected cells provides fundamental insights into the immunology of vaccines and recovery from infections.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.

Phase I study of intraventricular infusions of autologous ex vivo expanded NK cells in children with recurrent medulloblastoma and ependymoma.

BACKGROUND: Recurrent pediatric medulloblastoma and ependymoma have a grim prognosis. We report a first-in-human, phase I study of intraventricular infusions of ex vivo expanded autologous natural killer (NK) cells in these tumors, with correlative studies. METHODS: Twelve patients were enrolled, 9 received protocol therapy up to 3 infusions weekly, in escalating doses from 3 × 106 to 3 × 108 NK cells/m2/infusion, for up to 3 cycles. Cerebrospinal fluid (CSF) was obtained for cellular profile, persistence, and phenotypic analysis of NK cells. Radiomic characterization on pretreatment MRI scans was performed in 7 patients, to develop a non-invasive imaging-based signature. RESULTS: Primary objectives of NK cell harvest, expansion, release, and safety of 112 intraventricular infusions of NK cells were achieved in all 9 patients. There were no dose-limiting toxicities. All patients showed progressive disease (PD), except 1 patient showed stable disease for one month at end of study follow-up. Another patient had transient radiographic response of the intraventricular tumor after 5 infusions of NK cell before progressing to PD. At higher dose levels, NK cells increased in the CSF during treatment with repetitive infusions (mean 11.6-fold). Frequent infusions of NK cells resulted in CSF pleocytosis. Radiomic signatures were profiled in 7 patients, evaluating ability to predict upfront radiographic changes, although they did not attain statistical significance. CONCLUSIONS: This study demonstrated feasibility of production and safety of intraventricular infusions of autologous NK cells. These findings support further investigation of locoregional NK cell infusions in children with brain malignancies.

Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.

Steering CAR T cells to distinguish friend from foe.

CD19-specific chimeric antigen receptor (CAR)+ T cells have demonstrated clinical efficacy and long-lasting remissions, concomitant with tolerable normal B-cell aplasia. However, many tumor-associated antigens (TAAs) are expressed on normal tissues, the destruction of which would lead to intolerable toxicity. Thus, there is a need to engineer CAR+ T cells with improved safety profiles to restrict toxicity against TAA-expressing normal tissues. Bioengineering approaches include: (i) targeting CAR+ T cells to the tumor site, (ii) limiting CAR+ T-cell persistence, and (iii) restricting CAR activation. We review and evaluate strategies to engineer CAR+ T cells to reduce the potential of on-target, off-tissue toxicity.

Regulatable interleukin-12 gene therapy in patients with recurrent high-grade glioma: Results of a phase 1 trial.

Human interleukin-12 (hIL-12) is a cytokine with anticancer activity, but its systemic application is limited by toxic inflammatory responses. We assessed the safety and biological effects of an hIL-12 gene, transcriptionally regulated by an oral activator. A multicenter phase 1 dose-escalation trial (NCT02026271) treated 31 patients undergoing resection of recurrent high-grade glioma. Resection cavity walls were injected (day 0) with a fixed dose of the hIL-12 vector (Ad-RTS-hIL-12). The oral activator for hIL-12, veledimex (VDX), was administered preoperatively (assaying blood-brain barrier penetration) and postoperatively (measuring hIL-12 transcriptional regulation). Cohorts received 10 to 40 mg of VDX before and after Ad-RTS-hIL-12. Dose-related increases in VDX, IL-12, and interferon-γ (IFN-γ) were observed in peripheral blood, with about 40% VDX tumor penetration. Frequency and severity of adverse events, including cytokine release syndrome, correlated with VDX dose, reversing promptly upon discontinuation. VDX (20 mg) had superior drug compliance and 12.7 months median overall survival (mOS) at mean follow-up of 13.1 months. Concurrent corticosteroids negatively affected survival: In patients cumulatively receiving >20 mg versus ≤20 mg of dexamethasone (days 0 to 14), mOS was 6.4 and 16.7 months, respectively, in all patients and 6.4 and 17.8 months, respectively, in the 20-mg VDX cohort. Re-resection in five of five patients with suspected recurrence after Ad-RTS-hIL-12 revealed mostly pseudoprogression with increased tumor-infiltrating lymphocytes producing IFN-γ and programmed cell death protein 1 (PD-1). These inflammatory infiltrates support an immunological antitumor effect of hIL-12. This phase 1 trial showed acceptable tolerability of regulated hIL-12 with encouraging preliminary results.

Percutaneous BCG enhances innate effector antitumor cytotoxicity during treatment of bladder cancer: a translational clinical trial.

Background: Intravesical bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for treating patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, relapse rates remain high and BCG unresponsive NMIBC often requires bladder removal. Preclinical data suggest that priming with percutaneous BCG vaccine could improve response to intravesical BCG. Methods: A single-arm trial (NCT02326168) was performed to study the safety, immunogenicity, and preliminary efficacy of priming. Percutaneous BCG was given 21 days prior to intravesical BCG instillation in patients (n = 13) with high-risk NMIBC. Immune responses were monitored and compared to a sequentially enrolled cohort of nine control patients receiving only intravesical BCG. The effect of BCG on natural killer (NK) and γδ T cell in vitro cytotoxicity was tested. γδ T cell subsets were determined by T cell receptor gene expression with NanoString. Results: Priming was well tolerated and caused no grade ≥3 adverse events. The 3-month disease-free rate for prime patients was 85% (target goal ≥ 75%). Priming boosted BCG-specific immunity at 3 months and increased the activation status of in vitro expanded circulating NK and γδ T cells and their cytotoxicity against bladder cancer cells through receptor NKG2D. BCG enhanced the cytotoxicity of NK and γδ T cells against K562, RT4, and UM-UC6 but not against T24, UM-UC-3, or UM-UC-14 cells. Infiltrating γδ T cell subsets identified in the bladder includes γ9δ2 and γ8δ2. Conclusions: BCG priming is safe and tolerable. Poor sensitivity to NK and γδ T cell cytotoxicity by some bladder tumors represents a potential BCG-resistance mechanism.

Regulated intratumoral expression of IL-12 using a RheoSwitch Therapeutic System® (RTS®) gene switch as gene therapy for the treatment of glioma.

The purpose of this study was to determine if localized delivery of IL-12 encoded by a replication-incompetent adenoviral vector engineered to express IL-12 via a RheoSwitch Therapeutic System® (RTS®) gene switch (Ad-RTS-IL-12) administered intratumorally which is inducibly controlled by the oral activator veledimex is an effective approach for glioma therapy. Mice bearing 5-10-day-old intracranial GL-261 gliomas were intratumorally administered Ad-RTS-mIL-12 in which IL-12 protein expression is tightly controlled by the activator ligand, veledimex. Local tumor viral vector levels concomitant with veledimex levels, IL-12-mRNA expression, local and systemic cytokine expression, tumor and systemic flow cytometry and overall survival were studied. Ad-RTS-mIL-12+veledimex elicited a dose-related increase in tumor IL-12 mRNA and IL-12 protein and discontinuation of veledimex resulted in a return to baseline levels. These changes correlated with local immune and antitumor responses. Veledimex crossed the blood-brain barrier in both orthotopic GL-261 mice and cynomolgus monkeys. We have demonstrated that this therapy induced localized controlled production of IL-12 which correlates with an increase in tumor-infiltrating lymphocytes (TILs) leading to the desired biologic response of tumor growth inhibition and regression. At day 85 (study termination), 65% of the animals that received veledimex at 10 or 30 mg/m2/day were alive and tumor free. In contrast, the median survival for the other groups were: vehicle 23 days, bevacizumab 20 days, temozolomide 33 days and anti-PD-1 37 days. These findings suggest that the controlled intratumoral production of IL-12 induces local immune cell infiltration and improved survival in glioma, thereby demonstrating that this novel regulated immunotherapeutic approach may be an effective form of therapy for glioma.

Preclinical Efficacy and Safety of CD19CAR Cytokine-Induced Killer Cells Transfected with Sleeping Beauty Transposon for the Treatment of Acute Lymphoblastic Leukemia.

Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expand on those findings by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with the Sleeping Beauty (SB) transposon CD19CAR (CARCIK-CD19). Specifically, thanks to a large-scale 18-day manufacturing process, it was possible to achieve stable CD19CAR expression (62.425 ± 6.399%) and efficient T-cell expansion (23.36 ± 3.00-fold). Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in an established patient-derived xenograft (PDX) of MLL-ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a biodistribution and toxicity model. The infused cells persisted in the hematopoietic and post-injection perfused organs until the end of the study and consisted of CD8+, CD56+, and CAR+ T cells. Overall, these findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL, a possibility that will be tested in Phase I/II clinical trials after allogeneic hematopoietic stem-cell transplantation.

Etiologies and Impact of Readmission Rates in the First 180 Days After Hematopoietic Stem Cell Transplantation in Children, Adolescents, and Young Adults.

INTRODUCTION: High rates of patients require readmission to the hospital within 6 months of hematopoietic stem cell transplantation (HSCT). We investigated the relationship between readmission rates and outcomes after HSCT in children, adolescents, and young adults (CAYA). MATERIALS AND METHODS: A retrospective analysis of patients (26 years or younger) treated with HSCT was conducted. RESULTS: A chart review of 435 CAYA who underwent HSCT from 2008 to 2015 revealed that 171 patients (39%) had at least 1 hospital readmission within 180 days of transplant; 87% received allogeneic and 13% received autologous HSCT. A total of 312 readmission events were reported. The median follow-up time was 31 months. Documented infection (n=99) and graft-versus-host disease complications (n=60) were the most common causes. Higher than 2 readmission rates were associated with lower overall survival (OS) (P=0.001) and disease-free survival (P<0.001) in patients who received allogeneic HSCT. These findings were not found in the autologous HSCT. In a multivariate analysis of those who received allogeneic HSCT, prior treatment with ≥2 chemotherapy regimens (P=0.03) was independent predictor of lower OS. There were also trends noted toward lower OS for patients with documented infections at index admission or subsequent readmissions (P=0.09). CONCLUSIONS: More than 2 hospital readmissions within 6 months of allogeneic HSCT in CAYA, who are either heavily pretreated or had documented infections at index admission or subsequent readmissions adversely affected the outcomes.

Outcomes of children, adolescents, and young adults following allogeneic stem cell transplantation for secondary acute myeloid leukemia and myelodysplastic syndromes-The MD Anderson Cancer Center experience.

We conducted a retrospective analysis of outcomes for children and young adults with sAML/sMDS who underwent HSCT at our institution. Thirty-two patients (median age 20 years) with sAML (n=24) and sMDS (n=8) received HSCT between 1990 and 2013. The median time from sAML/sMDS diagnosis to HSCT was 4.1 months (range: 1.2-27.2 months). The transplant regimens were primarily busulfan based (n=19). BM was the primary donor source (n=15). Eleven recipients were transplanted with residual disease. At a median follow-up of 62.3 months (range: 0.4-250.9 months), 14 patients had disease recurrence. Acute GVHD, grade III/IV, occurred in three patients. Causes of death were as follows: disease relapse (n=12), infection (n=2), pneumonia (n=1), pulmonary hemorrhage (n=1), acute GVHD (n=1), and graft failure (n=1). A PS of ≥90% at the time of HSCT had a significant impact on PFS (P=.02). Patients achieving pretransplant primary CR (n=8) and those with sMDS and RA (n=6) had prolonged PFS (P=.04). On multivariate analysis, shorter time to transplantation (≤6 months from diagnosis of sAML/sMDS) was associated with superior OS (P=.0018) and PFS (P=.0005).

Targeting B-cell malignancies through human B-cell receptor specific CD4+ T cells.

The B-cell receptor (BCR) expressed by a clonal B cell tumor is a tumor specific antigen (idiotype). However, the T-cell epitopes within human BCRs which stimulate protective immunity still lack detailed characterization. In this study, we identified 17 BCR peptide-specific CD4+ T-cell epitopes derived from BCR heavy and light chain variable region sequences. Detailed analysis revealed these CD4+ T-cell epitopes stimulated normal donors' and patients' Th1 CD4+ T cells to directly recognize the autologous tumors by secretion of IFNγ, indicating the epitopes are processed and presented by tumor cells. One BCR peptide-specific CD4+ T cell line was also cytotoxic and lysed autologous tumor cells through the perforin pathway. Sequence analysis of the epitopes revealed that 10 were shared by multiple primary patients' tumors, and 16 had the capacity to bind to more than one HLA DRB1 allele. T cells stimulated by shared epitopes recognized primary tumors expressing the same sequences on multiple HLA DRB1 alleles. In conclusion, we identified 17 BCR-derived CD4+ T-cell epitopes with promiscuous HLA DRB1 binding affinity that are shared by up to 36% of patients, suggesting a strategy to overcome the requirement for individual preparation of therapeutic agents targeting idiotype.

Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells.

BACKGROUND: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS: SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS: CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION: Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING: National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.