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.

Multidimensional single-cell analysis identifies a role for CD2-CD58 interactions in clinical antitumor T cell responses.

The in vivo persistence of adoptively transferred T cells is predictive of antitumor response. Identifying functional properties of infused T cells that lead to in vivo persistence and tumor eradication has remained elusive. We profiled CD19-specific chimeric antigen receptor (CAR) T cells as the infusion products used to treat large B cell lymphomas using high-throughput single-cell technologies based on time-lapse imaging microscopy in nanowell grids (TIMING), which integrates killing, cytokine secretion, and transcriptional profiling. Our results show that the directional migration of CD19-specific CAR T cells is correlated with multifunctionality. We showed that CD2 on T cells is associated with directional migration and that the interaction between CD2 on T cells and CD58 on lymphoma cells accelerates killing and serial killing. Consistent with this, we observed that elevated CD58 expression on pretreatment tumor samples in patients with relapsed or refractory large B cell lymphomas treated with CD19-specific CAR T cell therapy was associated with complete clinical response and survival. These results highlight the importance of studying dynamic T cell-tumor cell interactions in identifying optimal antitumor responses.

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

Understanding immune response to infections and vaccines lags understanding humoral responses. While neutralizing antibody responses wane over time, T cells are instrumental in long-term immunity. We apply machine learning and time-lapse imaging microscopy in nanowell grids (TIMING) to study thousands of videos of T cells with specificity for SARS-CoV-2 eliminating targets bearing spike protein as a surrogate for viral infection. The data on effector functions, including cytokine secretion and cytotoxicity, provide the first direct evidence that cytotoxic T lymphocytes from a convalescent patient targeting an epitope conserved across all known variants of concern are serial killers capable of eliminating multiple infected target cells. These data have implications for vaccine development and for the recovery and monitoring of infected individuals.

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.

Combined immunotherapy with controlled interleukin-12 gene therapy and immune checkpoint blockade in recurrent glioblastoma: An open-label, multi-institutional phase I trial.

BACKGROUND: Veledimex (VDX)-regulatable interleukin-12 (IL-12) gene therapy in recurrent glioblastoma (rGBM) was reported to show tumor infiltration of CD8+ T cells, encouraging survival, but also up-regulation of immune checkpoint signaling, providing the rationale for a combination trial with immune checkpoint inhibition. METHODS: An open-label, multi-institutional, dose-escalation phase I trial in rGBM subjects (NCT03636477) accrued 21 subjects in 3 dose-escalating cohorts: (1) neoadjuvant then ongoing nivolumab (1mg/kg) and VDX (10 mg) (n = 3); (2) neoadjuvant then ongoing nivolumab (3 mg/kg) and VDX (10 mg) (n = 3); and (3) neoadjuvant then ongoing nivolumab (3 mg/kg) and VDX (20 mg) (n = 15). Nivolumab was administered 7 (±3) days before resection of the rGBM followed by peritumoral injection of IL-12 gene therapy. VDX was administered 3 hours before and then for 14 days after surgery. Nivolumab was administered every two weeks after surgery. RESULTS: Toxicities of the combination were comparable to IL-12 gene monotherapy and were predictable, dose-related, and reversible upon withholding doses of VDX and/or nivolumab. VDX plasma pharmacokinetics demonstrate a dose-response relationship with effective brain tumor tissue VDX penetration and production of IL-12. IL-12 levels in serum peaked in all subjects at about Day 3 after surgery. Tumor IFNγ increased in post-treatment biopsies. Median overall survival (mOS) for VDX 10 mg with nivolumab was 16.9 months and for all subjects was 9.8 months. CONCLUSION: The safety of this combination immunotherapy was established and has led to an ongoing phase II clinical trial of immune checkpoint blockade with controlled IL-12 gene therapy (NCT04006119).

Adoptive immunotherapy with double-bright (CD56bright /CD16bright ) expanded natural killer cells in patients with relapsed or refractory acute myeloid leukaemia: a proof-of-concept study.

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.

Designed improvement to T-cell immunotherapy by multidimensional single cell profiling.

BACKGROUND: Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. METHODS: To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. RESULTS: With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. CONCLUSIONS: Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

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.

Curvature sensing with a D-shaped multicore fibre and Brillouin optical time-domain reflectometry.

A distributed curvature sensor based on Brillouin optical time-domain reflectometry interrogation technique in a D-shaped 7-core fibre is presented. By comparing the relative Brillouin frequency shift between the central core and three of the outer cores of the 7-core fibre, the curvature of various spools with different diameters is measured with a deviation from the actual value ranging between 9% and 15%. The analysis and results presented in this study show the first demonstration of distributed bend sensing using a specially designed multicore D-shaped fibre, paving the way for fully distributed 3D shape sensing.

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.

Defining potency of CAR+ T cells: Fast and furious or slow and steady.

Genetically engineered T cells that express chimeric antigen receptors (CAR+) are heterogeneous and thus, understanding the immunotherapeutic efficacy remains a challenge in adoptive cell therapy. We developed a high-throughput single-cell methodology, Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to monitor interactions between immune cells and tumor cells in vitro. Using TIMING we demonstrated that CD4+ CAR+ T cells participate in multi-killing and benefit from improved resistance to activation induced cell death in comparison to CD8+ CAR+ T cells. For both subsets of cells, effector cell fate at the single-cell level was dependent on functional activation through multiple tumor cells.

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.

Human LAP+GARP+FOXP3+ regulatory T cells attenuate xenogeneic graft versus host disease.

Adoptive transfer of regulatory T cells (FOXP3+ Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after ex vivo expansion. Methods: We isolated bona fide Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR Vß diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their in vitro and in vivo potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. Results: LAP+ repurification results in >90% LAP+FOXP3+ Tregs, leaving behind FOXP3- and FOXP3+ nonTregs within the LAP- population. After 4-week expansion, the LAP+ Tregs were >1 billion cells, highly suppressive and anergic in vitro, >90% demethylated in the TSDR and able to maintain TCR Vß diversity. In the xGVHD model, exogenous CD25-PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP+ Tregs increased median survival to 47 days, while the LAP parent (CD25+) and LAP- nonTregs had median survival of 39 and 31 days, respectively. Conclusions: These preclinical data together provide evidence that LAP+ Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP+ Tregs to prevent or treat GVHD and other autoimmune diseases.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.