anti-GD2 Chimeric Antigen Receptor Research Articles

Comparative Study of the Effect of Radiation Delivered by Lutetium-177 or Actinium-225 on Anti-GD2 Chimeric Antigen Receptor T Cell Viability and Functions.

Chimeric antigen receptor (CAR) T cells have been relatively ineffective against solid tumors. Low-dose radiation which can be delivered to multiple sites of metastases by targeted radionuclide therapy (TRT) can elicit immunostimulatory effects. However, TRT has never been combined with CAR T cells against solid tumors in a clinical setting. This study investigated the effects of radiation delivered by Lutetium-177 (177Lu) and Actinium-225 (225Ac) on the viability and effector function of CAR T cells in vitro to evaluate the feasibility of such therapeutic combinations. After the irradiation of anti-GD2 CAR T cells with various doses of radiation delivered by 177Lu or 225Ac, their viability and cytotoxic activity against GD2-expressing human CHLA-20 neuroblastoma and melanoma M21 cells were determined by flow cytometry. The expression of the exhaustion marker PD-1, activation marker CD69 and the activating receptor NKG2D was measured on the irradiated anti-GD2 CAR T cells. Both 177Lu and 225Ac displayed a dose-dependent toxicity on anti-GD2 CAR T cells. However, radiation enhanced the cytotoxic activity of these CAR T cells against CHLA-20 and M21 irrespective of the dose tested and the type of radionuclide. No significant changes in the expression of PD-1, CD69 and NKG2D was noted on the CAR T cells following irradiation. Given a lower CAR T cell viability at equal doses and an enhancement of cytotoxic activity irrespective of the radionuclide type, 177Lu-based TRT may be preferred over 225Ac-based TRT when evaluating a potential synergism between these therapies in vivo against solid tumors.

P07.07.B INDUCTION AND TARGETING OF DIFFUSE MIDLINE GLIOMA IN A NOVEL HUMAN PONTINE ORGANOID MODEL

Abstract BACKGROUND Diffuse Midline Glioma (DMG) is a rare and aggressive pediatric brain cancer with no chance of survival, highlighting a critical need for therapy development. Disease representative models can aid the search for effective treatments, but human preclinical models that reflect the unique developmental features and anatomical environment of DMG, are so far lacking. Latest research on DMG suggest a fetal neurodevelopmental origin with a stem cell-like cellular profile. Here, we developed a novel pontine hindbrain model to create de novo H3K27M DMG and applied it to study early tumorigenesis and immunotherapy response. MATERIAL AND METHODS Timely sequenced morphogens were applied to developing cerebral organoids, to create a new pontine patterned brain organoid model. Bulk sequencing and 3D imaging over a period of 16 weeks were used to determine ideal patterning conditions, confirm the correct brain regionality and reconstruct cellular developmental dynamics. Introducing H3K27M with common pontine mutations P53 and PDGFRA-D842V via electroporation resulted in de novo H3K27M DMG, which subsequently was characterized based on WHO-histopathological criteria and single cell sequencing. Exploiting this new human DMG model, we applied barcode-based genetic lineage tracing paired with single cell sequencing, to delineate tumorigenesis, and anti-GD2 chimeric antigen receptor (CAR) T cell therapy to investigate CAR T cell responses. RESULTS Our novel pontine hindbrain organoid model inherently gives rise to all relevant macroglia and pons-specific neurons, resembling the same developmental dynamics as seen in humans. Moreover, de novo DMG introduced in these pontine organoids strongly resembles patient cancer, including its cellular human-specific heterogeneity and invasive nature, outperforming existing gold standard PDX and cell line models. As a first model for investigating DMG, the applied barcoded lineage-tracing delineates cancerous transforming precursor states and how they contribute to the diverse lineage among known DMG cancer cell populations. Finally, administration of CAR T cells recreates treatment outcomes observed in patients and demonstrates a high level of CAR T cell heterogeneity. CONCLUSION We generated a bona fide DMG pontine hindbrain organoid model, the first human in vitro model for this specific region of the brain. The matching resemblance to patient tumor paired with the relevant healthy brain environment gives this model the potential for new insights into DMG early tumorigenesis and microenvironmental impact, as well as next generation therapy development.

Anti-tumor efficacy of anti-GD2 CAR NK-92 cells in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are rare and fatal pediatric brainstem gliomas with no cure. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have been proven effective in treating glioblastoma (GBM) in preclinical studies. However, there are no relevant studies on the CAR-NK treatment for DIPG. Our study is the first to evaluate the anti-tumor activity and safety of GD2-CAR NK-92 cells treatment for DIPG. Five patient-derived DIPG cells and primary pontine neural progenitor cell (PPC) were used to access disialoganglioside GD2 expression. Cell killing activity of GD2-CAR NK-92 cells was analyzed by in vitro cytotoxicity assays. Two DIPG patient-derived xenograft models were established to detect the anti-tumor efficacy of GD2-CAR NK-92 cells in vivo. Among the five patient-derived DIPG cells, four had high GD2 expression, and one had low GD2 expression. In in vitro assays, GD2-CAR NK-92 cells could effectively kill DIPG cells with high GD2 expression while having limited activity against DIPG cells with low GD2 expression. In in vivo assays, GD2-CAR NK-92 cells could inhibit tumor growth in TT150630 DIPG patient-derived xenograft mice (high GD2 expression) and prolong the overall survival of the mice. However, GD2-CAR NK-92 showed limited anti-tumor activity for TT190326DIPG patient-derived xenograft mice (low GD2 expression). Our study demonstrates the potential and safety of GD2-CAR NK-92 cells for adoptive immunotherapy of DIPG. The safety and anti-tumor effect of this therapy need to be further demonstrated in future clinical trials.

Generation of anti-GD2 CAR macrophages from human pluripotent stem cells for cancer immunotherapies.

Macrophages armed with chimeric antigen receptors (CARs) provide a potent new option for treating solid tumors. However, genetic engineering and scalable production of somatic macrophages remains significant challenges. Here, we used CRISPR-Cas9 gene editing methods to integrate an anti-GD2 CAR into the AAVS1 locus of human pluripotent stem cells (hPSCs). We then established a serum- and feeder-free differentiation protocol for generating CAR macrophages (CAR-Ms) through arterial endothelial-to-hematopoietic transition (EHT). CAR-M produced by this method displayed a potent cytotoxic activity against GD2-expressing neuroblastoma and melanoma invitro and neuroblastoma invivo. This study provides a new platform for the efficient generation of off-the-shelf CAR-Ms for antitumor immunotherapy.

Highly Parallel Discovery of Synthetic Knockin Sequences for Enhanced Cancer Immunotherapies

Introduction T cells engineered to express transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged as powerful treatment options for some malignancies. However, while CAR T cells have induced impressive initial response rates in patients with B-precursor acute lymphoblastic leukemia (ALL) and lymphoma, they fail to mediate long-term relapse-free survival in 40-60% of patients and have not been successful in most solid tumors. For both TCR- and CAR-based approaches, T cell persistence and long-term functionality can be diminished by chronic antigen stimulation or tonic signaling. One approach to enhance the efficacy of cell therapies that has been explored by several groups is the overexpression of specific transcription factors (e.g. AP-1/ATF transcription factors such as c-JUN or BATF); however, more systematic gain-of-function screens in human CAR T cells have not yet been done. Methods and Results Here, we introduce CRISPR-based modular pooled knockin (ModPoKI) screens to evaluate hundreds to thousands of different constructs in human T cells and their potential to overcome TCR or CAR T cell dysfunction. We generated two barcoded ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs) to identify those that confer a fitness advantage to TCR or CAR T cells. The libraries were combined with either an NY-ESO-1-specific TCR, an anti-CD19 CAR or a high affinity anti-GD2 CAR that is known to induce tonic signaling-based dysfunction. The libraries and the TCR/CAR were engineered as polycistronic sequences that were non-virally integrated into the TRAC (T cell receptor alpha chain constant) locus of primary human T cells. We performed >20 unique ModPoKI screens including bead-based single stimulation, repetitive stimulation with target cells and tonic stimulation (GD2 CAR model). While constructs containing known AP-1/ATF transcription factors BATF and BATF3 showed increased abundance across multiple screens, transcription factor AP4 (TFAP4) knockin (KI) constructs were more clearly enriched after repetitive stimulation and tonic signaling, suggesting potentially undescribed benefits in exhaustion-prone environments. We next validated single knockins of TFAP4 or a control construct (truncated nerve growth factor receptor, tNGFR) in combination with the GD2 or the CD19 CAR. TFAP4 KI led to increased levels of cytokine release, proliferation and target cell killing compared to conventional CAR T cells. Moreover, TFAP4 KI mediated increased tumor control and survival in an in vivo model. Interestingly, we observed elevated levels of CD25 (IL2RA) expression on TFAP4 KI T cells and enrichment of genes related to IL2/STAT5 pathways which could hint at a potential benefit in situations of IL-2 competition/presence of regulatory T cells. Lastly, we aimed to discover specific combinations of TFs that work in concert to enhance T cell fitness in the setting of tonic CAR signaling. We created a ~10,000-member library (102 x 102 - pairwise combinations of 100 transcription factors plus two controls) cloned into constructs with the GD2 CAR and performed a tonic signaling ModPoKI screen. Analysis of the constructs that increased the most in relative abundance highlighted that several of the top performing constructs included either TFAP4, BATF, BATF3 or a combination of TFAP4 and BATF(3), suggesting that TFAP4 and BATF(3) are key transcription factors that can coordinately drive increased T cell fitness upon repetitive stimulation. Validation analyses confirmed that the combinatorial KI of a BATF-TFAP4 construct confers highest cytotoxic capacity in vitro and in vivo compared to single knockin (BATF-RFP or RFP-TFAP4) or control knockin (RFP-tNGFR) GD2 CAR constructs. Conclusions In conclusion, ModPoKI screens allow for customizable parallel evaluation and functional characterization of hundreds to thousands of different T cell constructs. Using clinically relevant screening modalities, we nominated candidate genes that can synthetically re-write the T cell response to repetitive antigen exposure or tonic signaling and thus have the potential to program more effective TCR or CAR T cell therapies for cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18.

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T cells redirected for universal cytokine-mediated killing”), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described “all-in-one” lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

BackgroundEwing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. MethodsThe anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. FindingsIn vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. InterpretationWe here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.

Anti-GD2 chimeric antigen receptor & trail modified mesenchymal progenitors as novel strategy against ewing’s sarcoma

Anti-GD2 chimeric antigen receptor & trail modified mesenchymal progenitors as novel strategy against ewing’s sarcoma

5P Anti-GD2 chimeric antigen receptor & TRAIL modified mesenchymal progenitors as novel strategy against Ewing’s sarcoma

5P Anti-GD2 chimeric antigen receptor & TRAIL modified mesenchymal progenitors as novel strategy against Ewing’s sarcoma

Design and Characterization of an "All-in-One" Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines.

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T cells redirected for universal cytokine-mediated killing”), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular “all-in-one” vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the GD2-specific CAR via GD2+ target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the “all-in-one” vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2+ tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy.

GD2-targeted chimeric antigen receptor T cells prevent metastasis formation by elimination of breast cancer stem-like cells

ABSTRACT Expression of the disialoganglioside GD2 has been identified as a marker antigen associated with a breast cancer stem-like cell (BCSC) phenotype. Here, we report on the evaluation of GD2 as a BCSC-specific target antigen for immunotherapy. GD2 expression was confirmed at variable degree in a set of breast cancer cell lines, predominantly in triple-negative breast cancer (TNBC). To target GD2, we have generated novel anti-GD2 chimeric antigen receptors (GD2-CAR), based on single-chain variable fragments (scFv) derived from the monoclonal antibody (mAb) ch14.18, also known as dinutuximab beta. Expressed on T cells, GD2-CARs mediated specific GD2-dependent T-cell activation and target cell lysis. In contrast to previously described GD2-CARs, no signs of exhaustion by tonic signaling were found. Importantly, application of GD2-CAR expressing T cells (GD2-CAR-T) in an orthotopic xenograft model of TNBC (MDA-MB-231) halted local tumor progression and completely prevented lung metastasis formation. In line with the BCSC model, GD2 expression was only found in a subpopulation (4-6%) of MDA-MB-231 cells before injection. Significant expansion of GD2-CAR-T in tumor-bearing mice as well as T-cell infiltrates in the primary tumor and the lungs were found, indicating site-specific activation of GD2-CAR-T. Our data strongly support previous findings of GD2 as a BCSC-associated antigen. GD2-targeted immunotherapies have been extensively studied in human. In conclusion, GD2-CAR-T should be considered a promising novel approach for GD2-positive breast cancer, especially to eliminate disseminated tumor cells and prevent metastasis formation.

Imaging CAR T Cell Trafficking with eDHFR as a PET Reporter Gene.

Cell-based therapeutics have considerable promise across diverse medical specialties; however, reliable human imaging of the distribution and trafficking of genetically engineered cells remains a challenge. We developed positron emission tomography (PET) probes based on the small-molecule antibiotic trimethoprim (TMP) that can be used to image the expression of the Escherichia coli dihydrofolate reductase enzyme (eDHFR) and tested the ability of [18F]-TMP, a fluorine-18 probe, to image primary human chimeric antigen receptor (CAR) Tcells expressing the PET reporter gene eDHFR, yellow fluorescent protein (YFP), and Renilla luciferase (rLuc). Engineered Tcells showed an approximately 50-fold increased bioluminescent imaging signal and 10-fold increased [18F]-TMP uptake compared to controls invitro. eDHFR-expressing anti-GD2 CAR Tcells were then injected into mice bearing control GD2- and GD2+ tumors. PET/computed tomography (CT) images acquired on days 7 and 13 demonstrated early residency of CAR Tcells in the spleen followed by on-target redistribution to the GD2+ tumors. This was corroborated by autoradiography and anti-human CD8 immunohistochemistry. We found a high sensitivity of detection for identifying tumor-infiltrating CD8 CAR Tcells, ∼11,000 cells per mm3. These data suggest that the [18F]-TMP/eDHFR PET pair offers important advantages that could better allow investigators to monitor immune cell trafficking to tumors in patients.

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.

Abstract PR04: GD2-directed chimeric antigen receptor T cells mediate potent antitumor effect and cure in xenograft models of diffuse intrinsic pontine glioma

Abstract Introduction: Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brainstem tumor with a median survival of less than one year. Despite advances in the understanding of the molecular origins of DIPG, improvement in clinical outcomes has yet to materialize. To date, there has been little target exploration for immunotherapy applications in DIPG. Methods: Patient-derived DIPG cell cultures were screened for expression of more than 350 surface antigens as potential immunotherapeutic targets. The disialoganglioside GD2 was found to have the highest expression across cell cultures and was verified by IHC on post-mortem samples. Chimeric antigen receptor (CAR) T-cell therapy against this target was explored both in vitro and in vivo. Results: We found high levels of the disialoganglioside GD2 expressed on cell cultures derived from post-mortem samples of DIPG. Quantification of the number of GD2 molecules per cell demonstrated higher GD2 expression on DIPG than any other tumors, including neuroblastoma, for which GD2 targeted immunotherapy is part of the standard of care. Most cases of DIPG are caused by a mutation in Histone 3.3 (H3K27M). GD2 is highly and uniformly expressed in patient-derived H3K27M DIPG cultures, whereas H3 wild-type pediatric high-grade gliomas, including those diagnosed as DIPG, do not express significant levels of GD2. The H3K27M mutation is associated with increased levels of enzymes in the ganglioside synthesis pathway, suggesting that expression of the target antigen is driven by H3K27M-induced transcriptional dysregulation. Anti-GD2 CAR T cells with a 4-1BB costimulatory domain demonstrate remarkable preclinical activity against H3K27M DIPG. GD2 CAR T cells specifically kill DIPG cells and produce cytokines IL-2 and IFN- upon coculture with tumor. Systemic administration of anti-GD2 CAR T cells achieves potent and durable cure compared to control T cells in multiple orthotopic xenograft models of DIPG. Using a CAR fluorescent protein fusion construct, we demonstrate significant T-cell trafficking to the brainstem where the antitumor effect is mediated. Universal response was observed across multiple cohorts, and treatment-associated toxicity was transient and tolerated during the period of peak antitumor activity. Conclusion: We have previously demonstrated that antigen density drives CAR efficacy. Extremely high expression of GD2 on DIPG makes this a particularly good disease for CAR T-cell therapy. If these results are predictive of human response, CAR T cells could have a transformative impact upon DIPG outcomes. A clinical trial of second generation anti-GD2 CAR T cells in relapsed and progressive DIPG is planned. Citation Format: Robbie G. Majzner, Christopher Mount, Shree Sundaresh, Evan Arnold, Meena Kadapakkam, Louai Labanieh, Pamelyn Woo, Michelle Monje, Crystal L. Mackall. GD2-directed chimeric antigen receptor T cells mediate potent antitumor effect and cure in xenograft models of diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR04.

Abstract 958: Anti-GD2 chimeric antigen receptor T cells as a potent immunotherapy regimen in xenograft models of histone 3 K27M mutant diffuse midline glioma

Abstract Histone 3 K27M mutated diffuse midline glioma (H3K27M DMG) is a universally fatal pediatric brain tumor. Despite improved understanding of the molecular origins of this disease, translations to improvement in clinical outcomes have yet to materialize. To date, there has been little target exploration for immunotherapy applications in H3K27M DMG. Here we report substantial preclinical efficacy of chimeric antigen receptor (CAR)-bearing T cells targeting the disialoganglioside GD2 in patient-derived orthotopic xenograft models of H3K27M DMG. GD2 is highly and uniformly expressed in patient-derived H3K27M DMG cultures, and in vitro assays demonstrate substantial target-dependent cytokine generation and target cell killing. Two H3 WT pediatric high-grade gliomas (HGG) cultures do not express significant levels of GD2, and the H3K27M mutation is associated with increased synthesis of ganglioside pathway synthesis enzymes, suggesting expression of the GD2 antigen is driven by H3K27M-induced transcriptional dysregulation. Single-dose systemic administration of GD2-4-1BBz-CAR T cells in multiple orthotopic xenograft models of H3K27M DMG achieves potent and lasting antitumor efficacy within a 4-week period of administration compared to a CD19-4-1BBz-CAR T cell control cohort. In brainstem xenografts, GD2-CAR T cell therapy cleared established tumor as measured by in vivo bioluminescence imaging and follow-up histology, and significantly prolonged survival. Using a fluorescently-labeled GD2-CAR, we demonstrate tumoricidal GD2-CAR T cells infiltrating the brain parenchyma and sparing of local neurons during tumor clearing. Treatment-associated neuroinflammation in brainstem xenografts is accompanied by transient ventriculomegaly due to swelling of the targeted tumors, which is accompanied by transient weight loss followed by full recovery. Similar swelling in thalamic xenograft models is lethal in a significant fraction of animals, presumably due to uncal herniation. These results demonstrate potent antitumor effects of GD2-directed CAR T cells in H3K27M DMG, and suggest an important role for neuroanatomic site in neurotoxicity associated with CAR T cell therapy. If these results are predictive of human response, GD2-directed CAR T cell therapy in the setting of careful clinical management could have a transformative impact upon H3K27M DMG outcomes. Citation Format: Christopher W. Mount, Robbie Majzner, Shree Sundaresh, Evan P. Arnold, Meena Kadapakkam, Samuel Haile, Louai Labanieh, Pamelyn Woo, Skyler P. Rietberg, Hannes Vogel, Michelle Monje, Crystal L. Mackall. Anti-GD2 chimeric antigen receptor T cells as a potent immunotherapy regimen in xenograft models of histone 3 K27M mutant diffuse midline glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 958.

Abstract CT145: A Cancer Research UK phase I trial of anti-GD2 chimeric antigen receptor (CAR) transduced T-cells (1RG-CART) in patients with relapsed or refractory neuroblastoma

Abstract Background: Treatment of high risk neuroblastoma remains challenging; current multimodal treatment regimens achieve long term survival in <50% of patients and are associated with significant morbidity. Ganglioside GD2 is abundantly expressed on almost all neuroblastomas whilst expression on normal tissue is highly limited, providing a suitable CAR target. Here, we report the preliminary results of a Phase I clinical study of GD2-CART for refractory/relapsed neuroblastoma (NCT02761915). Trial design: The therapeutic (1RG-CART) is autologous T-cells transduced with a gamma-retroviral vector encoding both an anti-GD2 CAR and the RQR8 suicide gene. The CAR comprises a humanized anti-GD2 single chain variable fragment derived from the K666 antibody and CD28/CD3ζ signalling domains. Both lymphodepletion and CART dose were escalated as follows: dose level (DL) 1 without lymphodepletion, DL2 with 1.2 g/m2 cyclophosphamide and DL3 and beyond 1.2 g/m2 cyclophosphamide and 125 mg/m2 fludarabine followed by administration of a single intravenous dose of 1x107/m2 (DL1-3) or 1x108/m2 (DL4) 1RG-CART. Primary objectives are to assess safety and tolerability. Results: To date, 12 patients have been enrolled. All had relapsed/refractory neuroblastoma with measurable disease in bone (n=11), bone marrow (n=7) and/or soft tissue sites (n=9). Cell products were successfully manufactured for all patients. Median transduction efficiency was 34.5% (range 16-54%). Nine patients have been treated on DL1 (n=4), DL2 (n=1), DL3 (n=1) and DL4 (n=3) respectively. No dose limiting toxicity (DLT) was seen. For patients treated on DL1-3 (1x107/m2), 1RG-CART could not be detected in peripheral blood, and no clinical responses were seen. In contrast, expansion of 1RG-CART cells as detected by flow cytometry and qPCR was seen in the 3 patients treated on DL4 (1x108/m2). In two DL4 patients, 1RG-CART expansion was still limited and transient (marking levels <10,000 copies/μg DNA). These patients had disease progression as measured at Day +28. In one DL4 patient however, 1RG-CART marking levels of >40,000 copies/μg DNA were achieved. This patient developed Grade 2 cytokine release syndrome (Day +5) and biochemical evidence of tumour lysis (Day +21). Disease reassessment on Day +28 showed response in many sites of bone/marrow disease as measured by mIBG scintigraphy, and near complete tumour clearance in bone marrow which at baseline was heavily infiltrated with neuroblastoma. Disease progression occurred on Day +45 at which time 1RG-CART were no longer detectable. In the absence of DLT this prompted us to continue with DL5 (1x109/m2). Conclusions: These preliminary results are the first to demonstrate on-target activity in bone and bone marrow of GD2-CART in this childhood solid tumour. Further 1RG-CART dose escalation is warranted, and under way. Citation Format: Karin Straathof, Barry Flutter, Rebecca Wallace, Simon Thomas, Gordon Cheung, Angela Collura, Talia Gileadi, Jack Barton, Gary Wright, Sarah Inglott, David Edwards, Claire Barton, Karen Dyer, Nigel Westwood, Thalia Loka, Sarita Depani, Karen Howe, Giuseppe Barone, Martin Pule, John Anderson. A Cancer Research UK phase I trial of anti-GD2 chimeric antigen receptor (CAR) transduced T-cells (1RG-CART) in patients with relapsed or refractory neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT145.

Abstract LB-111: Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion

Abstract In patients with chronic viral infection or cancer, continuous antigen exposure results in T cell exhaustion, which is characterized by sustained co-expression of multiple inhibitory receptors (ex. PD-1, TIM-3, LAG-3) and a hierarchical loss of effector function. Human T cells expressing a high-affinity anti-GD2 chimeric antigen receptor (CAR, HA.28z) develop phenotypic and functional hallmarks of exhaustion due to clustering of surface CAR and tonic signaling in the absence of antigen. In the present study, we exploited the HA.28z CAR as a model to characterize human T cell exhaustion and assess its potential for reversibility. CyTOF analyses of dysfunctional HA.28z CAR T cells demonstrated a distinct phenotype in which canonical exhaustion markers, T cell differentiation markers, and effector transcription factors were differentially expressed compared to CD19.28z CAR T cells that do not tonically signal. To interrogate the reversibility of T cell exhaustion, we engineered a regulatable HA.28z CAR by fusing a destabilization domain to the C-terminus of the CAR that rapidly induces CAR protein degradation. Addition of a small molecule stabilizes the protein and results in CAR surface expression. Using this model, we modulated the duration and intensity of tonic signaling by precisely modulating CAR surface expression. We hypothesized that transient cessation of tonic signaling would allow exhausted CAR T cells to recover and regain effector function. Following onset of exhaustion, elimination of CAR surface expression for 4 days resulted in a profound reversal of the exhausted phenotype. CyTOF analyses of reinvigorated cells indicated enhanced T cell memory formation, diminished surface marker expression of multiple exhaustion markers (ex. PD-1, TIM-3, LAG-3, CTLA-4, CD39) and reduced expression of transcription factors T-bet and Blimp-1. Furthermore, RNA sequencing of exhausted and reinvigorated HA.28z CAR T cells revealed a global molecular reprogramming upon removal of tonic CAR signaling, suggesting that CAR T cell exhaustion is functionally reversible. Upon CAR re-expression and co-culture with tumor antigen, reinvigorated HA.28z CAR T cells exhibited augmented killing and cytokine secretion compared to exhausted cells that continuously expressed surface CAR during expansion. In vivo studies assessing CAR T cell exhaustion reversibility using this model are currently being evaluated. In summary, tuning CAR surface expression offers a novel strategy to augment both the safety and efficacy of CAR T cell therapy. Moreover, these studies suggest that transient “rest” in T cells experiencing chronic antigen stimulation may be one mechanism by which exhaustion is prevented or reversed. Citation Format: Evan W. Weber, Rachel C. Lynn, Meena Malipatlolla, Elena Sotillo, Peng Xu, Crystal L. Mackall. Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-111.

Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)1-5 are aggressive and universally fatal pediatric brain cancers 6 . Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7-10, and recent results suggest benefit in central nervous system malignancies11-13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain 14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15-17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.

Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2

ABSTRACT Chimeric antigen receptors (CAR) and bispecific antibodies (BsAb) are two powerful immunotherapy approaches for retargeting lymphocytes toward cancer cells. Despite their success in lymphoblastic leukemia, solid tumors have been more recalcitrant. Identifying therapeutic barriers facing CAR-modified (CART) or BsAb-redirected T (BsAb-T) cells should facilitate their clinical translation to solid tumors. Novel lentiviral vectors containing low-affinity or high-affinity 4-1BB second-generation anti-GD2 (disialoganglioside) CARs were built to achieve efficient T cell transduction. The humanized anti-GD2 × CD3 BsAb using the IgG-scFv platform was described previously. CART and BsAb-engaged T cells were tested for viability, proliferation, and activation/exhaustion marker expression, and in vitro cytotoxicity against GD2(+) tumor cells. The antitumor effect of CAR-grafted and BsAb-T cells was compared in a human melanoma xenograft model. The majority of high CAR density T cells were depleted upon exposure to GD2(+) target cells while the BsAb-T cells survived. The in vitro cytotoxicity of the surviving CART cells was inferior to that of the BsAb-T cells. Using low-affinity CARs, inclusion of the 4-1BB co-stimulatory domain or exclusion of a co-stimulatory domain, or blocking PD1 did not prevent CART cell depletion. Both CART cells and BsAb-T cells penetrated established subcutaneous human melanoma xenografts; while both induced tumor regression, BsAb was more efficient. The fate of T cells activated by BsAb differs substantially from that by CAR, translating into a more robust antitumor effect both in vitro and in vivo.

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor

Chimeric antigen receptors (CARs) combine Tcell activation with antibody-mediated tumor antigen specificity, bypassing the need for Tcell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using Tcells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2+ Tcells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection.