Sonny Ang

Tuning Sensitivity of CAR to EGFR Density Limits Recognition of Normal Tissue While Maintaining Potent Antitumor Activity

Many tumors overexpress tumor-associated antigens relative to normal tissue, such as EGFR. This limits targeting by human T cells modified to express chimeric antigen receptors (CAR) due to potential for deleterious recognition of normal cells. We sought to generate CAR(+) T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies that differ in affinity. T cells with low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high-affinity cetuximab-CAR was not affected by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from nonmalignant cells.

Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

Frigault, Lee, and colleagues compared chimeric antigen receptors (CAR) encoding signaling domains comprising CD28, ICOS, and 4-1BB and found that some CD28 CAR-T cells have antigen-independent constitutive proliferation and cytokine secretion when highly expressed, leading to inferior antitumor effects. This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. Cancer Immunol Res; 3(4); 356–67. ©2015 AACR.

Sleeping beauty system to redirect T-cell specificity for human applications

The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19+ artificial antigen presenting cells was used to numerically expand CD3+ T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3+ T cells expressed CAR. CAR+ T cells specifically killed CD19+ target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR+ T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR V&agr; and TCR V&bgr; repertoire. Quantitative fluorescence in situ hybridization revealed that CAR+ T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR+ T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19+ B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).

Activating and Propagating Polyclonal Gamma Delta T Cells with Broad Specificity for Malignancies

Purpose: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. Experimental Design: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. Results: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts. Conclusions: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

Genetic editing of HLA expression in hematopoietic stem cells to broaden their human application.

Mismatch of human leukocyte antigens (HLA) adversely impacts the outcome of patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). This translates into the clinical requirement to timely identify suitable HLA-matched donors which in turn curtails the chances of recipients, especially those from a racial minority, to successfully undergo alloHSCT. We thus sought to broaden the existing pool of registered unrelated donors based on analysis that eliminating the expression of the HLA-A increases the chance for finding a donor matched at HLA-B, -C, and -DRB1 regardless of a patient’s race. Elimination of HLA-A expression in HSC was achieved using artificial zinc finger nucleases designed to target HLA-A alleles. Significantly, these engineered HSCs maintain their ability to engraft and reconstitute hematopoiesis in immunocompromised mice. This introduced loss of HLA-A expression decreases the need to recruit large number of donors to match with potential recipients and has particular importance for patients whose HLA repertoire is under-represented in the current donor pool. Furthermore, the genetic engineering of stem cells provides a translational approach to HLA-match a limited number of third-party donors with a wide number of recipients.

Antitumor activity of CD56-chimeric antigen receptor T cells in neuroblastoma and SCLC models

The CD56 antigen (NCAM-1) is highly expressed on several malignancies with neuronal or neuroendocrine differentiation, including small-cell lung cancer and neuroblastoma, tumor types for which new therapeutic options are needed. We hypothesized that CD56-specific chimeric antigen receptor (CAR) T cells could target and eliminate CD56-positive malignancies. Sleeping Beauty transposon-generated CD56R-CAR T cells exhibited αβT-cell receptors, released antitumor cytokines upon co-culture with CD56+ tumor targets, demonstrated a lack of fratricide, and expression of cytolytic function in the presence of CD56+ stimulation. The CD56R-CAR+ T cells are capable of killing CD56+ neuroblastoma, glioma, and SCLC tumor cells in in vitro co-cultures and when tested against CD56+ human xenograft neuroblastoma models and SCLC models, CD56R-CAR+ T cells were able to inhibit tumor growth in vivo. These results indicate that CD56-CARs merit further investigation as a potential treatment for CD56+ malignancies.

212. Of States and Fates: Predicting T-Cell Immunity By the Numbers

The immune system is a complex network of checks and balances in constant flux. Quantitative characterization of the system dynamics, sampled via constituent components such as T cells, combined with mathematical modeling enabled us to obtain “statistical pattern recognition” of immune states and transitions over time. Here we show a statistical framework to characterize immune states for adoptive immunotherapy using serial infusions of activated polyclonal T cells into companion canines diagnosed with B-cell non-Hodgkin Lymphoma (NHL) post CHOP chemotherapy regimen as a model for human disease. We applied multiplexed gene profiling techniques to assess changes in gene expression data from 10 companion canine patient clinical samples and gene regulatory networks (GRN) information to build an “immune landscape” that predicts immune states, state transitions, and plasticity for immunomodulation (“reprogrammability”) over the course of immunotherapy regimens. The potency of immune modulation and immune surveillance, two critical parameters of efficacy through inferred state transitions, were evaluated by measuring distortions and shifts in the immune landscape of canine patients undergoing treatment through system dynamics, Adjusted Rand Index, Pearson Correlation, and ANOVA analyses. Adjust Rand Index validated hierarchical clustering analyses. Pearson correlations isolated genes that were up- or down-regulated with regard to single genes of interest. ANOVA analyses indicated gene expression difference between temporal samples. Applying such analyses to adoptive T-cell immunotherapy, allowed us to examine clinical/tumor remissions through the lens of immune state transitions, affording real-time improvements to therapeutic regimens. In developing a formal framework to capture the effects of molecular signatures and GRN over time to describe distinct immune states, we enhanced the clinical cancer immunologists analytical arsenal (with a measuring tool/sensor) and helped bridge the gap between bench and bedside.

208. Single-Cell Gene Expression Profiles of Genetically Modified T Cells for Adoptive Immunotherapy

System-level characterization of cell states requires detailed portrayal of cellular components such as gene transcripts or proteins. Here, we exploit a robust single-cell platform technology to characterize T cells expanded ex vivo intended for human application. Coupled with advanced custom analytics, we identify “drivers” (relatively few, but vital) genes which orchestrate T-cell activation and clonal expansion programming, versus “passengers” (relatively many, less critical) or resultant/downstream transcripts.Detailed analyses reveal that there is considerable variability between individual activated T cells, implying that cell state may undergo multiple stepwise transitions in a stochastic manner. Compared to interrogation of bulk T-cell populations (e.g., analyzed using RNA-Seq, microarrays, and Northern blotting), where heterogeneity in expression signals is attenuated by temporal and cell-cycle averaging, the genetic heterogeneity between cells in isogenic populations may be a reflection of fundamental phenomena such as transcriptional bursting. Our model describes cell state oscillation in tandem with transcriptional repressive (closed chromatin) and permissive conformations (open chromatin), versus simpler probabilistic models of transcription. T-cell variability can significantly impact upon individual cell behavior within seemly isogenic populations, and may be essential in molding survival and cytotoxicity within certain contexts, such as the rapidly changing and stressful tumor microenvironments. We find that lactate dehydrogenase A (LDHA) is a leading indicator for the metabolic adaptation during T-cell activation and together with other energetics related genes such pyruvate kinase isoform 2 (PKM2), can be harnessed to adjust our culture conditions during ex vivo expansion. These cellular fingerprints suggest useful ways for tuning the balance between oxidative and non-oxidative glucose metabolism to enhance the killing and thus therapeutic potential of our T cells. Our framework to harness catalogs of gene expression data into clinically-applicable insights can be adapted for meaningful characterization of other therapeutically-relevant source populations such as hematopoietic stem cells and reprogrammed NK cells. This is being reduced to practice by understanding the heterogeneity within and between genetically modified T-cell products generated for clinical use.

226. Next-Generation Sleeping Beauty Integration Profiles of T-Cell Adoptive Immunotherapy

Since the first human Gene Therapy clinical trial was initiated 25 years ago, many technological advances have dove-tailed into the field, moving personalized genetic treatments from concept towards routine reality. In particular, widespread adoption of next-generation sequencing technologies have enabled faster vector integration profiling to assay for potential genotoxities. Genomic data generation since 2007 has grown faster than Moores law for computing power, now making data analysis the new bottleneck in the workflow. Newer and accelerated solutions are needed to manage exponential increases in data accumulation. Our goal is to compress sequence data generation and downstream data analysis within 5 work days, enabling the utility of genotoxicity assay to support time-sensitive clinical decision making. Time and cost savings in integrated and streamlined data processing should translate into easier and faster implementation in clinical settings. We use non-viral transfections, based on the Sleeping Beauty transposition system, to reprogram T cells for Adoptive Immunotherapy. We exploit the fact that Sleeping Beauty transposons invariably inserts into “TA” sites via a cut-and-paste mechanism driven by cognate transposases, to develop an analysis platform, SBhashmap, to quickly locate and unlock insights associated with the genomic integration sites. We chose hash tables over other table data structures for speed, an advantage which is more apparent when the number of entries is large. SBhashmap consists of pre-populated hash tables with over 150 million entries corresponding to the “TA” sites from the human genome and hash functions to match for quick site matching. By cataloging the TA sites ahead of time, we can cut the mapping time significantly. To benchmark SBhashmap, we compared transposon integration mapping to BLAST and BLAT matching for human genomic sequences, and found a 20-fold reduction in processing time, while maintaining similar levels of accuracy. This kind of hash maps is particularly efficient because the maximum number of entries is known in advance, such that the bucket array can be allocated once with the optimum size fixed (not resized, with no insertions and deletions necessary). With collision-free hash functions, the keys are not stored in the tables. With minor adaptations, SBhashmap can process genomic data from experimentally relevant model organisms such mice, worms, and fruitflies.