Activity Of Chimeric Antigen Receptor Research Articles

CD22 CAR Optimization for Improved in-Human Activity Following Inadequate CD22 CAR Activity in Phase 1 Clinical Trial PLAT-04

Background: CD19 targeting chimeric antigen receptor (CAR) T cells have induced unprecedented remission rates in high-risk precursor B Acute Lymphoblastic Leukemia (ALL); however recurrent disease with CD19 antigen escape variants is not uncommon. Therefore, we developed a novel CD22 targeting CAR, and following preclinical validation, tested it in a first-in-human pediatric and young adult phase 1 clinical trial, PLAT-04 (NCT03244306). Four subjects were treated at 2 dose levels (DL) (1x10 6/kg (DL1) and 3x10 6/kg (DL2)). The CD22 CAR T cell product (SCRI-CAR22v1) was successfully manufactured (n=4) and no dose limiting toxicity (DLTs), cytokine release syndrome (CRS) or neurotoxicity was observed. However, all subjects had minimal CAR T cell expansion, with 3 of 4 subjects demonstrating persistent or progressive disease at day 21 evaluation despite continued CD22 expression on leukemic blasts. Based on the poor in vivo expansion and lack of activity, enrollment was voluntarily halted to interrogate and optimize the CAR construct for enhanced performance.Methods: Human T cells were transduced to express one of two CD22 CAR constructs. We designed SCRI-CAR22v2, a CD22 CAR that utilizes the same scFv as SCRI-CAR22v1 but with a shorter linker between M971 VH and VL and a shorter hinge with differing transmembrane region, and both using CD8 alpha (Figure A). This construct maintained the truncated EGFR extracellular tag (EGFRt) for tracking and potential in vivo suicide mechanism. The two transduced CAR T cell products were compared preclinically by flow cytometry, chromium release assay and in an in vivo murine model to understand differing T cell activity between the CAR constructs. Additionally, SCRI-CAR22v2 is currently under investigation in a dose finding phase 1 clinical trial, PLAT-07 (NCT04571138).Results: Following use of cetuximab-APC and biotinylated anti-human Fab antibody for surface EGFRt and CAR detection, the SCRI-CAR22v1 expresses lower levels of EGFRt but similar CAR levels on the cell surface demonstrated by MFI (Figure B). Biotinylated, soluble CD22 antigen was also used to evaluate CD22 CAR receptor activity and, as measured by MFI, a higher affinity is suggested via SCRI-CAR22v2 as compared to SCRI-CAR22v1 (Figure B). K562 cells expressing low, medium or high CD22 were used to evaluate the impact of surface antigen expression on the CAR activity level. SCRI-CAR22v2 demonstrates improved targeted cell lysis at all 3 antigen quantity levels by chromium release assay (Figure C). In NSG mice inoculated with Raji tumor cells expressing ffluc, SCRI-CAR22v2 demonstrated improved survival compared to SCRI-CAR22v1 (Figure D) and clearance of Raji tumor cells (Figure E). Based on this promising preclinical data, we initiated enrollment onto PLAT-07, a phase 1 dose finding trial (2x10 5cells/kg (DL1), 5x10 5cells/kg (DL2) and 1x10 6cells/kg (DL3)) of SCRI-CAR22v2. To date, 3 subjects have been enrolled and successfully infused at DL1. All had prior CD19-CAR therapy and 2 lacked CD19 leukemic expression at the time of SCRI-CAR22v2 infusion. At the time of cell infusion, one subject had only extramedullary disease, one had MRD of <1% and one subject had a larger disease burden of 30% ALL. None experienced a DLT and all were MRD negative in the bone marrow at day 28 and the subject with EMD demonstrated a complete metabolic response by PET scan. Figure F exhibits the improved expansion and engraftment of the SCRI-CAR22v2 cells as compared to SCRI-CAR22v1 DL1 (n=3) and DL2 (n=1), and higher peak levels of CD22 CAR T cells as compared to SCRI-CAR22v1 DL1 and DL2 (Figure G).Conclusions: Despite encouraging preclinical data, SCRI-CAR22v1 demonstrated poor expansion and engraftment in a Phase 1 trial. Notably, minor CAR alterations lead to encouraging in-human activity in early clinical findings. Our experience suggests a shorter linker and hinge as well as incorporation of an CD8 alpha transmembrane region improves the clinical activity of CD22 targeted CAR T cells in subjects with recurrent disease following CD19 CAR T cells. Further evaluation is needed to elucidate the critical CAR components and/or assays at the preclinical level that can best predict which CAR should be brought to the clinic for further evaluation. [Display omitted] DisclosuresOrentas: Lentigen: Patents & Royalties. Jensen: BMS: Patents & Royalties; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Research Funding. Gardner: Novartis: Consultancy; BMS: Patents & Royalties.

Dexamethasone Enhanced CAR T Cell Persistence and Function through Upregulation of Interleukin 7 Receptor

Dexamethasone (Dex) has been a mainstay for the treatment of inflammatory pathologies, such as in autoimmunity and cytokine release syndrome (CRS) from immunotherapies. However, its effects on chimeric antigen receptor (CAR) T cells, during CRS events have not been interrogated. In this study, we treated the CAR T cells with different concentrations of Dex (0, 1, and 10µM) single or multiple times in vitro and expanded them for one week. We demonstrated that Dex treatment did not inhibit CAR T cell growth and functionality even with concentrations higher than what would be used in the clinic. Interestingly, we observed that the Dex treatment significantly upregulated endogenous gamma chain cytokine receptor-interleukin 7 receptor alpha (IL7Rα) at the mRNA and protein levels (P=0.0005) (Fig 1a). These effects are not T cell subset dependent because we observed upregulation of IL7Rα on PBMC and enriched naïve and memory T cell-derived CAR T cells. Furthermore, un-transduced T cells also exhibited IL7Rα increase, which suggests that the upregulation of IL7Rα is the general mechanism of Dex for T cells. IL7Rα is well accepted as a key element to CAR T cell persistence and memory T cell formation. However, the IL7R-IL-7 signaling pathway is limited due to the downregulation of high-affinity IL7Rα during the activation and expansion of CAR T cells. We found out that Dex can upregulate endogenous IL7Rα in a reversible manner, which is an important factor for safety in clinical application. We showed that ex vivo upregulation of IL7Ra by a single Dex treatment subsequently enhanced CAR T cell persistence and anti-tumor efficacy in vivo in the presence of IL-7. To further confirm the positive effects of Dex on CAR T cell therapy, we performed a combinatorial therapy by delivering CD19 CAR T cells to acute lymphoid leukemia (ALL) tumor-bearing NOD-scid IL2Rgammanull (NSG) mice and then administering Dex (1mg/kg) and IL-7-expressing CHO cells. Consistently, we observed a complete cure of tumor-bearing mice only in the CD19 CAR T cell group that was given both Dex and IL-7, but not CAR alone and Dex only groups (Fig 1b-c) (P=0.0006). Mice survived, tumor-free, over 150 days. Supportively, we observed CAR T cell persistence only in the CAR T cells combined with Dex and IL7 group but not in the CAR group. To determine if Dex influenced CAR T cells beyond IL7Rα, we performed gene analysis and demonstrated that IL7Rα, but not other γ chain cytokines was selectively upregulated by Dex, which supports previous reports from Lee et al. that Dex and glucocorticoid receptors (GR) complex binds upstream of the IL-7rα promoter preferentially regulating IL7Rα. Furthermore, we utilized Nanostring technology to analyze CAR T cells with and without Dex treatment for mRNA signatures that related to signaling pathways. We observed pathways related to activation, migration, persistence, and chemokine production were upregulated, while pathways related to apoptosis and TCR diversity were downregulated after Dex treatment. The results indicated that Dex may regulate multiple functions of CAR T cells.Overall, our studies in both in vitro and in vivo treatment support that Dex does not have negative effects on CAR T cell potency but provides insight into an unforeseen strategy to improve CAR T cell therapies through upregulation of IL-7Rα and improving T cell activation, trafficking, and persistence. We believe our observations could extend beyond hematological malignancies to a potentially potent and durable therapy for solid tumors, as Dex is not only an immunosuppressive agent but also an anti-cancer drug used against a multitude of tumors to prevent tumor growth as well as modulate the microenvironment. Our data also provided rationale on starting CAR T cell therapy without the necessity of tapering off the ongoing steroid treatment. [Display omitted] DisclosuresForman: Allogene: Consultancy; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company. Wang: Pepromene Bio, Inc.: Consultancy.

Project Stella: Development and Preclinical Assessment of FOLR1-Directed Chimeric Antigen Receptor T Cells in CBF2AT3-GLIS2/RAM AML

Background: A rare but highly aggressive type of AML that is only seen in infants with a unique immunophenotype (RAM phenotype) is caused by cryptic CBFA2T3-GLIS2 (CBF/GLIS) fusion. This infant AML is highly refractory to conventional chemotherapy with near uniform fatality despite highly intensive and myeloablative therapy (PMID 23153540). Transcriptome profiling of CBF/GLIS AML has revealed new insights into the pathogenesis of the fusion and uncovered fusion-specific molecular biomarkers that could be used for risk stratification and to inform treatment (PMID 30592296). Studying the largest cohort of these high-risk infants, we demonstrated several alterations in gene expression and transcriptional networks in these CBF/GLIS-positive patient samples that have potential for therapeutic targeting (PMID 31719049). FOLR1, which encodes for folate receptor alpha, was highly and uniquely expressed in CBF/GLIS AML but was entirely absent in AML with other cytogenetics abnormalities and in normal hematopoietic cells. Furthermore, we recently demonstrated that forced expression of CBF/GLIS enhances the proliferation and alters differentiation in cord blood (CB) CD34+ early precursors towards megakaryocytic lineage that recapitulates acute megakaryocytic leukemia seen in infants (PMID 31719049). Of significance, we showed that FOLR1 surface expression is causally linked to CBF/GLIS-induced malignant transformation, thus making it an attractive antigen for targeted therapies against CBF/GLIS AML cells. Given that chimeric antigen receptor (CAR) T cells are extremely effective at eradicating relapsed/refractory B-ALL malignancies, we developed FOLR1-directed CAR T cells for pre-clinical evaluation in CBF/GLIS AML.Methods: We generated a FOLR1-directed CAR using anti-FOLR1 binder (Farletuzumab), IgG4 intermediate spacer and 41-BB/CD3zeta signaling domains. The pre-clinical efficacy of FOLR1 CAR T cells was evaluated against CBF/GLIS AML cell lines in vitro and in vivo. CBF/GLIS AML models include CB CD34+ cells transduced with CBF/GLIS expression construct (CBF/GLIS-CB) and WSU-AML cell line. We also engineered Kasumi-1 cell line to express FOLR1 (Kasumi-1 FOLR1+) to evaluate target specificity (Figure 1A).Results: We tested the target specificity of FOLR1-directed CAR T cells against FOLR1-positive (CBF/GLIS-CB, WSU-AML, Kasumi-1 FOLR1+) and FOLR1-negative (Kasumi-1) cells. CD8 FOLR1 CAR T cells demonstrated cytolytic activity against FOLR1 positive but not FOLR1 negative cells (Figure 1B). Furthermore, both CD8 and CD4 FOLR1 CAR T cells produced higher levels of IL-2, IFN-γ, and TNF-α and proliferated more robustly than did unmodified T cells when co-incubated with FOLR1 positive but not FOLR1 negative cells (Figure 1C). These results indicate highly specific reactivity of FOLR1 CAR T cells against AML cells expressing FOLR1. We next investigated the in vivo efficacy of FOLR1-directed CAR T cells. In CBF/GLIS-CB, WSU-AML, and Kasumi-1 FOLR1+ xenograft models, treatment with FOLR1 CAR T cells induced leukemia clearance, while disease progression occurred in all mice that received unmodified T cells (Figure 1D). Activity of FOLR1 CAR T cells in vivo was target specific, as they did not limit the leukemia progression nor extend the survival of Kasumi-1 xenografts (Figure 1D).To determine whether FOLR1 is expressed on normal HSPCs, we characterized FOLR1 expression in normal CB CD34+ samples. FOLR1 expression was entirely silent in HSPC subsets (Figure 1E). Consistent with lack of expression, no cytolytic activity was detected against HPSCs Moreover, FOLR1 CAR T cells did not affect the self-renewal and multilineage differentiation capacity of normal HSPCs as compared to unmodified control T cells (Figure 1F), whereas significant eradication of colonies were detected in the CBF/GLIS-CB cells (Figure 1G).Conclusion: In this study, we demonstrate that FOLR1 CAR T effectively eradicates CBF/GLIS AML cells without compromising normal HSPCs, providing a promising approach for the treatment of high-risk CBF/GLIS AML. Transition of this CAR T to clinical development for infant AML is underway. [Display omitted] DisclosuresHylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML.We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells.In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. DisclosuresPardo: Hematologics, Inc.: Current Employment.

Developing a Safer Anti-CD44v6 Chimeric Antigen Receptor T Cell Against Hematological Cancers By Mitigating on-Target Off-Tumor Toxicity

Adoptively transferred chimeric antigen receptor (CAR) T cells have shown significant promise in targeted immunotherapy against hematological tumors, despite concerns with safety and antigen escape. The CD44 adhesion molecule, which binds components of ECM such as collagen and hyaluronan, has been implicated in growth and survival as well as metastasis of tumor initiating stem cells (TSCs). A splice variant of CD44, CD44v6 is broadly expressed on malignant tumors including AML, CML and multiple myeloma (MM), where it aids tumor migration and predicts poor disease prognosis 1. Moreover, the expression of CD44v6 on healthy tissues including keratinocytes, presents a significant challenge in ‘on-target, off-tumor’ toxicity 2,3. We have developed a second generation, anti-CD44v6 CAR T cell (CD44v6CAR) that mediates potent cytotoxic and effector function against AML and MM in-vitro. Additionally, we observed lesser potency of our CD44v6CAR T cells against MM in comparison with AML in-vivo. Interestingly, higher levels of soluble CD44v6 antigen were also detected in mice engrafted with MM.1S compared to THP1. Possibly, the presence of soluble CD44v6 in mouse serum culminated in reduced anti-tumor activity against MM in-vivo, most likely due to CAR scFv blockade with antigen. Here, we provide an alternative strategy for improving the CD44v6 CAR T cell therapy and mitigating on-target off-tumor toxicity using CD44v6 CAR T cells that secrete a soluble protease-susceptible version of the CAR-expressed CD44v6 scFv that will block CAR binding in healthy tissue, but will be cleaved by cancer-specific proteases in the tumor site, allowing for CAR T cell binding and activation (Figure 1). We leveraged the presence of matrix metalloproteinase (MMP)-2, which is significantly overexpressed in multiple myeloma tumor microenvironment 4 to develop a modified CAR T construct (sCD44v6CAR) with engineered MMP-2 cut site in the linker between the heavy and light chains of the soluble CD44v6scFv. Kinetic analysis with varying MMP-2 concentration and digestion time showed moderate proteolytic susceptibility of our scFv construct, with digestion efficiency increasing in a dose-dependent manner. Furthermore, our engineered protease-liable scFv demonstrated higher affinity for CD44v6 antigen binding in titration assays compared to CAR scFv suggesting that engineered scFv are able to 1) bind and neutralize soluble antigen and 2) bind antigen on healthy tissues with high affinity to mitigate off-tumor toxicity. As expected, both conventional (CD44v6CAR) and modified sCD44v6 CAR T cells showed effective cytotoxicity against AML in vitro. Interestingly, cytotoxic activity against MM.1S using the modified T cells (sCD44v6CAR) was significantly suppressed, likely resulting from secretion of soluble scFv. The addition of recombinant MMP-2 in co-culture assays cleaved soluble scFvs, rescuing CAR-mediated tumor killing. Taken together, the data confirms our proof-of-concept hypothesis and highlights the protective capacity of engineered sCD44v6CAR T cells, with its ability to potentially neutralize off-target toxicity and improve anti-MM activity in future studies, which has impact on the CAR T cell therapy as a general strategy.Figure 1: Schematic representation of CAR T Cell mitigating off-disease recognition. Presence of protease-susceptible linker in soluble scFv is efficiently cleaved by tumor-specific proteases in tumor microenvironment enabling CAR binding and activation. Lack of specific proteases in healthy tissues leads to high affinity, soluble scFv-target binding and effective blocking.

Conditioned CAR-T cells by hypoxia-inducible transcription amplification (HiTA) system significantly enhances systemic safety and retains antitumor efficacy

BackgroundHypoxia is a striking feature of most solid tumors and could be used to discriminate tumors from normoxic tissues. Therefore, the design of hypoxia-conditioned Chimeric Antigen Receptor (CAR) T cells...

Titratable Pharmacological Regulation of CAR T Cells Using Zinc Finger-Based Transcription Factors.

Simple SummaryChimeric antigen receptor (CAR) T cell therapy can be associated with substantial side effects primarily due to intense immune activation following treatment, or target antigen recognition on off-tumor tissue. Consequently, temporal and tunable control of CAR T cell activity is of major importance for the clinical translation of innovative CAR designs. This work demonstrates the transcriptional regulation of an anti-CD20 CAR in primary T cells using a drug inducible zinc finger-based transcription factor. The switch system enables titratable induction of CAR expression and CAR T cell effector function with the clinically relevant inducer drug tamoxifen and its metabolites both in vitro and in vivo, whereby CAR activity is strictly dependent on the presence of the inducer drug. The results obtained can readily be transferred to other CARs for which an improved control of expression is required.Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

Recent Progress in Genome Editing for Gene Therapy Applications: The French Perspective.

Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.

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Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

Treating solid malignancies with chimeric antigen receptor (CAR) T cells typically results in poor responses. Immunomodulatory biologics delivered systemically can augment the cells' activity, but off-target toxicity narrows the therapeutic window. Here we show that the activity of intratumoural CAR T cells can be controlled photothermally via synthetic gene switches that trigger the expression of transgenes in response to mild temperature elevations (to 40-42 °C). In vitro, heating engineered primary human T cells for 15-30 min led to over 60-fold-higher expression of a reporter transgene without affecting the cells' proliferation, migration and cytotoxicity. In mice, CAR T cells photothermally heated via gold nanorods produced a transgene only within the tumours. In mouse models of adoptive transfer, the systemic delivery of CAR T cells followed by intratumoural production, under photothermal control, of an interleukin-15 superagonist or a bispecific T cell engager bearing an NKG2D receptor redirecting T cells against NKG2D ligands enhanced antitumour activity and mitigated antigen escape. Localized photothermal control of the activity of engineered T cells may enhance their safety and efficacy.

Remodeling of Tumor Microenvironment by Tumor-Targeting Nanozymes Enhances Immune Activation of CAR T Cells for Combination Therapy.

Targeting B7-H3 chimeric antigen receptor (CAR) T cells has antitumor potential for therapy of non-small cell lung cancer (NSCLC) in preclinical studies. However, CAR T cell therapy remains a formidable challenge for the treatment of solid tumors due to the heterogeneous and immunosuppressive tumor microenvironment (TME). Nanozymes exhibit merits modulating the immunosuppression of the tumor milieu. Here, a synergetic strategy by combination of nanozymes and CAR T cells in solid tumors is described. This nanozyme with dual photothermal-nanocatalytic properties is endowed to remodel TME by destroying its compact structure. It is found that the B7-H3 CAR T cells infused in mice engrafted with the NSCLC cells have superior antitumor activity after nanozyme ablation of the tumor. Importantly, it is found that the changes altered immune-hostile cancer environment, resulting in enhanced activation and infiltration of B7-H3 CAR T cells. The first evidence that the process of combination nanozyme therapyeffectively improves the therapeutic index of CAR T cells is presented. Thus, this study clearly supports that the TME-immunomodulated nanozyme is a promising tool to improve the therapeutic obstacles of CAR T cells against solid tumors.

Bispecific CAR T Cells against EpCAM and Inducible ICAM-1 Overcome Antigen Heterogeneity and Generate Superior Antitumor Responses.

Adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated unparalleled responses in hematologic cancers, yet antigen escape and tumor relapse occur frequently. CAR T-cell therapy for patients with solid tumors faces even greater challenges due to the immunosuppressive tumor environment and antigen heterogeneity. Here, we developed a bispecific CAR to simultaneously target epithelial cell adhesion molecule (EpCAM) and intercellular adhesion molecule 1 (ICAM-1) to overcome antigen escape and to improve the durability of tumor responses. ICAM-1 is an adhesion molecule inducible by inflammatory cytokines and elevated in many types of tumors. Our study demonstrates superior efficacy of bispecific CAR T cells compared with CAR T cells targeting a single primary antigen. Bispecific CAR T achieved more durable antitumor responses in tumor models with either homogenous or heterogenous expression of EpCAM. We also showed that the activation of CAR T cells against EpCAM in tumors led to upregulation of ICAM-1, which rendered tumors more susceptible to ICAM-1 targeting by bispecific CAR T cells. Our strategy of additional targeting of ICAM-1 may have broad applications in augmenting the activity of CAR T cells against primary tumor antigens that are prone to antigen loss or downregulation.

Abstract 1541: Incorporation of an IgG4-derived spacer improves the activity of nanobody-based CAR T cells targeting a membrane-distal epitope of glypican 1 in pancreatic cancer

Abstract Background &amp; Aims: Epitope recognition and antigen density have emerged as major parameters affecting the anti-tumor activity of chimeric antigen receptor (CAR) T cells. Glypican 1 (GPC1) is a cell surface proteoglycan that is expressed in pancreatic cancer. Here, we investigate how the choice of an epitope and an extracellular spacer in CAR design can determine T cell activation in high- or low- GPC1-expressing tumors. Methods: We use mouse hybridoma and phage display technologies to isolate GPC1-specific antibodies or nanobodies with distinct epitopes, and analyze GPC1 protein expression in pancreatic cancer. We construct CARs with different extracellular spacers and test them in GPC1-positive cell and mouse models. We use droplet digital PCR and genomic sequencing methods to analyze persistent CAR T cells in mice. Results: We isolate a camel nanobody D4 and a mouse monoclonal antibody HM2 recognizing the membrane-distal and membrane-proximal epitopes of GPC1, respectively, with similar high affinity. GPC1 protein was frequently detected in pancreatic tumor tissues and normal pancreas adjacent to tumors. Both D4 and HM2 CAR T cells with the hinge and transmembrane domain (TM) from CD8 regressed high GPC1-expressing xenograft tumors. Interestingly, D4 CAR containing a short IgG4 hinge spacer and CD28 TM outperformed the initial D4 CAR against low GPC1-expressing tumors in vitro and in vivo. Conclusion: By optimizing spacer and TM designs, the activity of nanobody D4-based CAR T cell targeting a membrane-distal epitope of GPC1 is significantly enhanced against low GPC1-expressing tumors, suggesting it might improve the efficacy of GPC1-targeted CAR T cell therapy in pancreatic cancer. Citation Format: Nan Li, Dan Li, Jiajia Pan, Mitchell Ho. Incorporation of an IgG4-derived spacer improves the activity of nanobody-based CAR T cells targeting a membrane-distal epitope of glypican 1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1541.

Abstract LB141: Assessment of natural killer (NK) cell activity for immunotherapy using a novel flow cytometry-based killing assay

Abstract Natural killer (NK) lymphocytes exhibit potent responses against transformed and pathogen-infected cells. NK cells can act rapidly as they monitor the bloodstream and tissues for tumorigenic and infected cells that lack “self” markers of MHC class I. Upon recognition of a foreign invader or tumor cell, NK cells release cytotoxic proteins such as interferon-gamma and granzyme B. Because of their ability to recognize stressed cells lacking “self” markers, human NK cells have shown promise as therapeutics in clinical trials to reduce disease burden in patients with therapy-resistant or advanced-stage blood cancers. With more than one hundred NK cell- and induced NK cell-based clinical trials ongoing for blood cancers and refractory solid tumors, there is an ever-increasing need for NK cell therapies to treat cancer and immune diseases. We sought to develop novel, clinically relevant, and robust methods for human NK cell expansion. In addition, to simplify the processing of cells for characterization, we developed a flow cytometry based killing assay to monitor NK activity. Therefore, within the same experiment, we can analyze NK cell phenotype profiles with specific antibody panels while also assessing NK cell killing activity. Using optimized serum- and xeno-free conditions with minimal cell manipulations, we have expanded highly purified NK cells up to 500-fold in 14 days from human peripheral blood mononuclear cells (PBMCs) using optimized cytokines and Cloudz CD2/NKp46 microspheres. To characterize the resulting NK cell population, we used a unique antibody panel to phenotype the NK cells by flow cytometry. Within the same experiment, NK killing assays against K562 cells can be performed using a flow cytometry-based killing assay, which has the advantage of using one instrument for all analyses, shortened processing times, and reproducibility. Using this workflow, we can identify different culture conditions that adversely affect NK activity. Together, this data presents a comprehensive and clinically relevant workflow for the expansion of highly purified NK cells and assessing their killing potential. Furthermore, this workflow can be used for screening the activity of chimeric antigen receptor (CAR) expressing immune cells against specific cancer antigens. Citation Format: Jody Bonnevier, Christine Goetz, Li Peng, Jamie Van Etten, Bora Faulkner, Christopher Hammerbeck, Ariel Miller, Gabriella Perell, Christopher Johnson, Joseph Lomakin, David Hermanson, Kevin Flynn. Assessment of natural killer (NK) cell activity for immunotherapy using a novel flow cytometry-based killing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB141.

Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer

Emerging data demonstrate that the activity of immune cells can be modulated by microbial molecules. Here, we show that the short-chain fatty acids (SCFAs) pentanoate and butyrate enhance the anti-tumor activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells through metabolic and epigenetic reprograming. We show that in vitro treatment of CTLs and CAR T cells with pentanoate and butyrate increases the function of mTOR as a central cellular metabolic sensor, and inhibits class I histone deacetylase activity. This reprogramming results in elevated production of effector molecules such as CD25, IFN-γ and TNF-α, and significantly enhances the anti-tumor activity of antigen-specific CTLs and ROR1-targeting CAR T cells in syngeneic murine melanoma and pancreatic cancer models. Our data shed light onto microbial molecules that may be used for enhancing cellular anti-tumor immunity. Collectively, we identify pentanoate and butyrate as two SCFAs with therapeutic utility in the context of cellular cancer immunotherapy.

Abstract LB105: In vivo CAR T therapy: targeted in vivo gene delivery of a CAR using a CD8-specific fusogen results in tumor eradication

Abstract CAR T cells are highly effective at inducing remissions in patients with refractory B cell malignancies. The ex vivo process used to manufacture these therapies limits patient access and exposes T cells to non-physiologic conditions in culture. Viral vectors are efficient at genetically modifying T cells and are used in the ex vivo manufacture of most CAR T cell products but lack target-cell specificity. Here we describe how viral envelope glycoproteins, termed fusogens, engineered to target the CD8 co-receptor can efficiently and specifically deliver a chimeric antigen receptor (CAR) to human T cells in vivo that support target cell killing and tumor eradication. Modification of viral envelope proteins to include antibody-based binding domains (CD8 binders) can produce fusogen-viral vector compositions that deliver genetic payloads to CD8 T cells with therapeutic effect (1, 2). Screening CD8-targeted binders identified fusogens with a range of on-target cell transduction efficiencies and off-target cell line specificities. CD8-targeted fusogens with the highest efficiency were comparable to VSVg-pseudotyped lentiviruses at transducing CD8 T cells, but unlike VSVg showed CD8 T cell specific transduction. Interestingly, CD8-targeted fusogens were superior to VSVg-pseudotyped lentivirus at transducing non-activated human T cells. In vivo fusogen-mediated delivery showed efficient and specific CD8 T cell transduction and expression of a GFP reporter gene. Second-generation CARs containing a CD19 binding domain and a 41BB costimulatory domain delivered by targeted fusogen demonstrated CD8 specific expression and functional activity against non-malignant B cells and CD19+ Nalm6 leukemia cells. To demonstrate targeted fusogen-mediated in vivo CAR delivery and activity, we established Nalm6 xenografts in mice into which unmodified activated human PBMCs were infused. Activated human PBMCs alone were unable to control tumor growth. A single intravenous delivery of a CD8 fusogen containing a second-generation CD19 CAR transgene across a range of functional titer doses resulted in the generation of CD8 CAR Ts that eradicated the CD19+ tumor xenografts. A high percentage of T cells demonstrated CAR expression after fusogen delivery with clear specificity for the CD8+ cells. Importantly, the fusogen was able to generate a functional CAR response regardless of prior activation status of the T cells. Targeted in vivo delivery of CAR into T cell subsets may represent a novel therapeutic approach for human B cell malignancies. The ability to specifically deliver a CAR gene to a T cell in vivo would be transformative in terms of patient access and is likely to generate a qualitatively superior therapeutic T cell.

A Direct Comparison of the In Vivo Efficacy and in Vitro Signaling of Chimeric Antigen Receptors and Endogenous T Cell Receptors

The adoptive transfer of chimeric antigen receptor (CAR) expressing T cells targeting the CD19 antigen induces remission in 70-90% of pediatric patients with relapsed and/or refractory acute lymphoblastic leukemia (ALL). Despite initial enthusiasm, emerging data indicate the durability of remission is limited in a substantial number of patients, therefore, addressing relapse will be critical for increasing the clinical efficacy of CAR T cell therapy. CARs are synthetic receptors designed to redirect the antigen specificity of T cells while recapitulating the T cell activating properties of the endogenous T cell receptor (TCR). Clinical and pre-clinical observations have demonstrated that CAR T cells have inferior sensitivity to low levels of antigen and have diminished persistence in vivo compared to T cells responding to TCR activation. These observations suggest the CAR does not fully replicate the signaling of a TCR which subsequently leads to suboptimal T cell function which may underlie the inability of CAR T cell therapy to induce durable remissions in a significant number of patients.In these studies, we directly compared the in vivo and in vitro consequences of CAR versus TCR activation, as well as the signal transduction of each of these receptors. To accomplish this, we created a novel, syngeneic model system in which a 2nd generation CAR is introduced into OTI TCR transgenic T cells to generate a population of T cells (CAR-OTI cells) in which each cell recognizes the CD19 antigen via its CAR as well as the ovalbumin (OVA) antigen via its TCR, thus providing a model in which the function and signaling of each receptor could be studied within a uniform population of T cells. To stimulate either the CAR or the TCR, we generated antigen-expressing cell lines from the pre-B ALL cell line, E2aPbx, that expressed either the cognate antigen for the CAR (CD19), the TCR (OVA) or both. These cell lines allowed us to stimulate either receptor in a model which preserved the properties of the cell-to-cell interactions expected in vivo . We validated the activity of each receptor in this model, by demonstrating similar in vitro degranulation, proliferation and target cell lysis in CAR-OTI cells upon co-incubation with CAR-antigen or TCR-antigen positive E2aPbx cells. Despite the similarities seen in vitro, CAR-OTI cells adoptively transferred into mice with established E2aPbx leukemia demonstrated enhanced expansion at 4 (p<0.001) and 6 days (p<0.01) post-infusion in TCR-activated CAR-OTI cells, relative to CAR-activated CAR-OTI cells. Likewise, clearance of leukemia from the bone marrow (p<0.001) and survival (p<0.01) were each superior in mice in which CAR-OTI cells were activated through the TCR, relative to the CAR. These results demonstrate the significant difference in in vivo behavior and efficacy of CAR- versus TCR-mediated T cell activation, and suggest these differences may result from differences in the signals generated by each receptor.Using our model of CAR-OTI cells and E2aPbx cells expressing either the CAR- or TCR-antigen, we interrogated proteins in the canonical TCR signal transduction cascade after CAR or TCR activation. We demonstrated that CAR activation resulted in increased and prolonged phosphorylation of Zap70, a kinase central to canonical TCR signal transduction. Despite the increased activation of Zap70, the CAR signal demonstrated diminished activation of the MAPK family members, Erk (p<0.001) and p38 (p<0.05), which correlated with the diminished in vivo efficacy seen in this model. Furthermore, this model was used to define the global phosphoproteome of CAR- and TCR-activated CAR-OTI cells by a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) methodology in conjunction with mass spectrometry (MS). Our SILAC-MS analysis demonstrated multiple differences in the signal transduction cascade of CARs relative to TCRs, including proteins of the signalosome as well as the NFAT and NF-kB pathways. These data demonstrate that a CAR generates an “altered” signal, relative to that of a TCR, correlating with diminished in vivo expansion and efficacy in the treatment of established leukemia. These findings underscore the need to better understand the biology of chimeric antigen receptors as a means of rationally altering their design to improve their clinical effectiveness in mediating durable remissions in patients with relapsed/refractory ALL. DisclosuresNo relevant conflicts of interest to declare.

Modulation of CD22 Antigen Density Improves Efficacy of CD22 Chimeric Antigen Receptor (CAR) T Cells Against CD22lo B-Lineage Leukemia and Lymphoma

While overall survival for acute lymphoblastic leukemia (ALL) in children treated with risk-adapted, multi-agent chemotherapeutic regimens is greater than 85%, relapsed ALL remains a major challenge. Chimeric antigen receptor (CAR) T cell therapy targeting CD19 provides these patients with a remission induction rate of 70-90% across multiple clinical trials, but loss of the targeted epitope results in relapse following CD19 CAR T cells. In the setting of these relapses, our group conducted a CD22 CAR T cell trial, which induces remission in approximately 70% percent of patients with relapsed/refractory CD22-positive pre-B cell ALL, including those relapsing after CD19 CAR therapy (NCT02315612). However, relapse associated with reduced CD22 site density has shortened remission duration in some patients. To test the impact of low CD22 site density on CD22 CAR T cell functionality, we modified Nalm6 ALL cell lines using CRISPR/Cas9 to eliminate the CD22 gene, followed by lentiviral transduction of full-length CD22 and single-cell cloning based on CD22 expression levels. CD22 site density was assessed with the Quantibrite-PE Assay Kit and four cell lines were identified for a spectrum of CD22 expression: CD22 negative (CD22neg); CD22 low (CD22lo) - site density comparable to that seen in ALL patients relapsing following CD22 CAR therapy; CD22 normal (Nalm6) - site density comparable to ALL CD22 expression; and CD22 high (CD22hi) - site density comparable to CD19 levels. As CD22 site density decreases, CD22 CAR T cells produce incrementally less interferon-gamma and do not produce IL-2 against CD22lo ALL. CD22 CAR delayed, but was unable to prevent, progression of CD22lo ALL in murine xenografts. We next evaluated two potential approaches to overcoming low site density antigen escape: improving CAR activity or increasing tumor sensitivity. To improve CAR activity, we generated a high-binding affinity CD22 CAR (CD22 CARH1) using an affinity-enhanced version of the scFv from the CD22 CAR construct in our ongoing trial. Despite higher affinity, the CD22 CARH1 T cell did not improve in vitro killing or in vivo clearance of CD22lo ALL. To increase tumor sensitivity, we sought to increase CD22 site density using the PKC inhibitor Bryostatin1, a drug previously shown to increase CD22 expression in CLL. Bryostatin1 increases CD22 site density on a panel of pre-B cell ALL and diffuse large B cell lymphoma (DLBCL) cell lines. Interestingly, the CD22 increase is durable for at least 1 week after exposure to Bryostatin1. Bryostatin1-mediated CD22 increased site density improves cytokine production by CD22 CAR T cells. To evaluate the mechanism of Bryostatin1-mediated upregulation of CD22 expression, RNAseq was performed on Bryostatin1-exposed ALL. Preliminary analysis demonstrates that Bryostatin1 does not directly affect CD22 mRNA expression levels, but modulates early transcription factors within the B-cell receptor pathway. Based on these results, we are further evaluating pathways affected by Bryostatin1 through protein expression analysis and assessing in vivo function of Bryostatin1 in the setting of a patient-derived CD22 CAR-resistant xenograft model. In conclusion, these results demonstrate a clear threshold antigen site density, below which the CD22 CAR is unable to clear CD22-expressing ALL. We further demonstrate that increased CD22 CAR affinity does not seem to improve CD22 CAR activity against CD22lo ALL. Bryostatin1-mediated CD22 modulation can improve CD22 CAR response against leukemia and lymphoma providing a potential rational combination to rescue CD22 CAR activity and remission durability. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting TGFβ Signaling in CAR T-Cells May Significantly Enhance Efficacy of Tumor Immunotherapy

The use of genetically engineered T cells expressing chimeric antigen receptors (CAR) against CD19 or BCMA to treat hematologic malignancies has demonstrated promising results in clinical trials. However, it is possible that the efficacy of CAR T cells could be limited in some clinical indications due to suppressive factors in the tumor microenvironment. Immunosuppressive cytokines (e.g., IL-6, IL-10 and TGFβ) provide a favorable environment for tumor growth and inhibit the tumoricidal activity of the endogenous T cells. In particular, transforming growth factor β (TGFβ) has been shown to suppress T-cell proliferation, inhibit the maturation of T helper cells and reduce their effector functions. Elevated levels of TGFβ have been detected in many human cancers, including multiple myeloma (MM). Here, we sought to modulate the activity of CAR T cell-based immunotherapy for MM by inhibiting TGFβ signaling in BCMA-specific CAR T cells (Budha et al., Cancer Cell, 2012).We evaluated two strategies to modulate the TGFβ pathway: (1) knocking out TGFβ Receptor II (TGFβRII) using CRISPR gene editing and (2) overexpressing a dominant negative TGFβRII in BCMA targeted CAR T cells. TGFβRII modulation inhibited TGFβ-dependent Smad2/3 phosphorylation. TGFβRII-modulated BCMA CAR T cells were insensitive to TGFβ-mediated inhibition of proliferation, cell cytotoxicity and cytokine production, both IFNg and granzyme B, in the context of primary and/or repeat antigen stimulation with BCMA+ve MM cell lines in vitro . We further evaluated the gene expression signatures of BCMA CAR T cells isolated from tumor and spleen in a NSG mouse xenograft model subcutaneously inoculated with myeloma cell line (RPMI8226). TGFβ pathway gene expression changes indicating TGFβ exposure were observed in BCMA CAR T cells isolated from tumor, but not spleen, suggesting that BCMA CAR T cells are encountering a TGFβ-enriched TME. However, both TGFβ pathway modulating strategies prevented the development of the TGFβ-induced gene expression phenotype.Together, these data suggest that inhibition of TGFβRII signaling may potentiate CAR T activity by directly inhibiting immunosuppressive effects of TGFβ in the tumor microenvironment. By specifically targeting TGFβ signaling in CAR T cells, it may be possible to avoid side effects associated with systemic administration of TGFβ antagonists. Gene editing to remove TGFβRII expression or overexpressing dominant negative TGFβ RII could modulate the activity of BCMA CAR T therapy in MM. DisclosuresVong:Juno Therapeutics: Employment, Equity Ownership. Nye:Juno Therapeutics: Employment, Equity Ownership. Hause:Juno Therapeutics: Employment, Equity Ownership. Clouser:Juno Therapeutics: Employment, Equity Ownership. Jones:Juno Therapeutics: Employment, Equity Ownership. Burleigh:Juno Therapeutics: Employment, Equity Ownership. Borges:Editas Medicine: Employment, Equity Ownership. Chin:Editas Medicine: Employment, Equity Ownership. Marco:Editas Medicine: Employment, Equity Ownership. Barrera:Editas Medicine: Employment, Equity Ownership. Da Silva:Editas Medicine: Employment, Equity Ownership. Harbinski:Editas Medicine: Employment, Equity Ownership. Giannoukos:Editas Medicine: Employment, Equity Ownership. Dhanapal:Editas Medicine: Employment, Equity Ownership. Jiang:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment, Equity Ownership. Wilson:Editas Medicine: Employment, Equity Ownership. Myer:Editas Medicine: Employment, Equity Ownership. Welstead:Editas Medicine: Employment, Equity Ownership. Bond:Juno Therapeutics: Employment, Equity Ownership. Sather:Juno Therapeutics: Employment, Equity Ownership.

CD19-Specific Chimeric Antigen Receptor-Modified T Cells with Safety Switch Produced Under “Point-of-Care” Using the Sleeping Beauty System for the Very Rapid Manufacture and Treatment of B-Cell Malignancies

Chimeric antigen receptor (CAR)-modified autologous T cells have shown remarkable clinical responses targeting B-cell malignancies. However, a majority of manufacturing approaches rely on expensive viral vectors and ex vivo activation of clinical-grade T cells for gene transfer with subsequent 2-3 week-long numeric expansion to achieve necessary dosing. We have engineered our clinically-validated Sleeping Beauty (SB) system for stable non-viral integration of CAR, cytokine signaling, and safety switch transgenes into T cells to eliminate need for viral vectors. We previously demonstrated that co-expression of membrane bound IL-15 (mbIL15, Proc Natl Acad Sci U S A. 2016 Nov 29;113(48):E7788-E7797) on CD19-specific CAR+ T cells using SB system allows for very rapid (<2 days) manufacturing with point-of-care (P-O-C) technology (Blood 2016 128:2807). This approach to production avoids the need for activation and propagation of CAR+ T cells leading to augmented persistence and anti-tumor activity in an in vivo model of leukemia. We now update this technology by co-expressing a safety switch, HER1t, on CD19-specific CAR+ mbIL15+ T cells. HER1t improves the safety profile by providing a mechanism for selective in vivo depletion of P-O-C-produced T cells through the administration of cetuximab, a clinically-available monoclonal antibody. We confirmed co-expression of CAR, mbIL15, and HER1t in P-O-C-manufactured T cells and demonstrated that these cells exhibited a memory-like phenotype, potent antigen-specific cytotoxicity, and produced IFNγ, TNFα, and IL-2 when co-cultured with CD19+ tumor cells. Cetuximab specifically and efficiently killed mbIL15+ HER1t+ CAR+ T cells via antibody dependent cellular cytotoxicity and complement dependent cytotoxicity in a dose-dependent manner. Adoptive transfer of mbIL15+HER1t+CAR+ T cell generated in less than 2 days under P-O-C into immunocompromised (NSG) mice bearing established CD19+ leukemia (NALM-6) resulted in significant reduction in tumor burden and improvement in overall survival, compared to control mice (Figure). Blood analysis demonstrated improved expansion and persistence of mbIL15+ HER1t+ CAR+ T cells compared to mbIL15negHER1tnegCAR+ T-cell control. In summary, we have generated T cells co-expressing mbIL15 and the HER1t safety switch in less than two days using P-O-C that demonstrated superior efficacy and persistence compared to P-O-C T cells that expressed CAR without mbIL15. These pre-clinical data demonstrate the effectiveness of very rapidly manufactured mbIL15+ HER1t+ CAR+ T cells in targeting CD19+ tumors and provide a rationale for clinical evaluation. [Display omitted] DisclosuresChan:Intrexon Corporation: Employment. Gallagher:Intrexon Corporation: Employment. Cheng:Intrexon Corporation: Employment. Carvajal-Borda:Intrexon Corporation: Employment. Plummer:Intrexon Corporation: Employment. Govekung:Intrexon Corporation: Employment. Barrett:ZIOPHARM Oncology: Employment. Khare:Ziopharm Oncology: Employment. Cooper:Immatics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Miltenyi Biotec: Honoraria; Ferring: Consultancy; Intrexon Corporation: Equity Ownership, Patents & Royalties; Argos Therapeutics: Equity Ownership; Targazyme, Inc.: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Research Funding; Organovo Holdings: Equity Ownership; Procter & Gamble: Equity Ownership; AmpliPhi Biosciences: Equity Ownership; Sangamo Biosciences: Patents & Royalties. Shah:Intrexon Corporation: Employment.