GD2 Expression Research Articles

Stability of the HSV-2 US-6 Gene in the del II, del III, CP77, and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells.

The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2) US6 gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the CP77 gene locus (MVA_009–MVA_013) and the I8R-G1L intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and CP77 sites. Sequencing analysis implicated US6 deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the I8R-G1L site, suggesting better accommodation of transgenes in this intergenic region. Thus, the I8R-G1L intergenic region may be more useful for transgene insertion for enhanced stability.

Abstract P3-02-05: Targeting glutamine metabolism inhibits GD2+ breast cancer stem cell function in triple negative breast cancer

Abstract Breast cancer stem cells (BSCs) constitute a fraction of primary tumor cells that exhibit drug resistance and have metastatic potential. Ganglioside GD2 has been shown by us and others as a marker for BCSCs. Nutrient deprivation-associated metabolic stress observed during tumor progression is reportedly associated with the cancer stem cell phenotype, and we have shown that oxidative stress caused by serum deprivation induces GD2 expression in vitro and in vivo. To identify metabolic signatures associated with GD2+ cells and their potential adaptive mechanisms to oxidative stress, global metabolic profiling was performed using a mass spectroscopy-based approach. We found that metabolites associated with amino acid metabolism, particularly glutathione metabolism, to be most highly upregulated in GD2+ compared to GD2− cells. In addition, glutathione biosynthesis and oxidation to glutathione disulfide was also increased in cells undergoing oxidative stress. These data suggest that the glutathione-mediated detoxification pathways play a key role in GD2+ or basal type breast cancer cells. As glutamine is a major precursor molecule for glutathione biosynthesis, we hypothesized that glutamine, at least in part, regulates cellular redox tone and the GD2+ phenotype in breast cancer cells. To test our hypothesis, we cultured breast cancer cell lines MDA-MB-231 and SUM159 in media containing 0%, 0.5%, 1%, 2%, or 4% of L-glutamine for 3 days. Flow cytometry analysis revealed that the percentage of GD2+ increased from 6% to 12% and from 12% to 22% in MDA-MB-231 and SUM159 cells, respectively when the cells were cultured at 0 or 4% glutamine. Glutaminase, an enzyme that converts glutamine to glutamate, which further leads to glutathione biosynthesis, has been shown to be overexpressed in basal-type breast cancer cells. To investigate the role of glutaminase on stem cell redox homeostasis and phenotype, we treated TNBC cell lines MDA-MB-231 and SUM159 cells with glutaminase inhibitors CB-839 or 968 compound for 3 days. Flow analysis revealed 70-80% reduction in GD2 expression after treatment in a dose dependent manner. In addition, treatment with CB-839 inhibited cell proliferation by over 80% in a dose-dependent manner in MDA-MB-231 cells and SUM159 cells (p<0.001). Next, to determine the effect of glutaminase inhibition on BCSC function, we cultured MDA-MB-231 and SUM-159 cells in low adherent cell culture dishes with or without glutaminase inhibitors 968 or CB-839 for 7 days. Interestingly, we observed a 40-50% reduction in mammosphere formation in both treated cell lines compared to the untreated controls, suggesting that inhibition of glutaminase is critical for BCSC function. In conclusion, the anti-oxidant glutathione is highly upregulated in GD2+ BCSCs, and this appears to be associated with the concentration of glutamine, and appears to regulate, perhaps via the regulation of redox homeostaisis, GD2 expression in BCSCs. Targeting glutaminase and redox homeostasis using CB-839 or CB968 inhibits BCSC as measured by GD2 positivity and mammosphere formation. Glutaminase inhibition in combination with chemotherapy could be a valuable future therapeutic strategy for targeting BCSCs in TNBC. Citation Format: Stanley Ly, Khoa Nguyen, Michael Andreeff, Venkata Lokesh Battula. Targeting glutamine metabolism inhibits GD2+ breast cancer stem cell function in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-05.

Profiling of O-acetylated Gangliosides Expressed in Neuroectoderm Derived Cells.

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.

Expression of ganglioside GD2 on colorectal adenocarcinoma cells

Using flow cytometry GD2 ganglioside expression was evaluated both on colorectal adenocarcinoma cell lines and on tumor tissue samples from colorectal cancer patients. The marker was found on EpCAM-positive tumor cells in 6 of 12 patients' samples but not on the HT29 and CaCo-2 cell lines. GD2 expression was not an exceptional feature of cancer stem cells, since its expression level was similar on CD133-positive and CD133-negative tumor cells. Thus, the presence of GD2 ganglioside was revealed on colorectal adenocarcinoma cells for the first time. This finding makes it possible to use targeted therapy to treat this disease.

PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

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.

CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BackgroundChimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects.MethodsCD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs.ResultsAll retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing.ConclusionBoth CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

Abstract 1169: Characterization of a small molecule inhibitor for GD3 synthase (ST8SIA1), a novel target in breast cancer stem-like cells

Abstract We reported that GD2 selectively identifies breast cancer stem-like cells (BCSCs) in TNBCs and that the enzyme ST8SIA1 regulates GD2 biosynthesis. We have shown that knockout of ST8SIA1 expression in TNBC cell lines inhibits tumor growth and metastasis in vivo, whereas ST8SIA1 overexpression induces epithelial-to-mesenchymal transition, leading to metastasis of TNBC cells. Very recently we have shown that ST8SIA1 regulates focal adhesion kinase (FAK)/AKT/mammalian target of rapamycin (mTOR) signaling in GD2+ BCSCs. Here we hypothesize that inhibition of ST8SIA1 activity using small molecule inhibitors targets BCSC function. Because of a lack of a crystal structure for developing small-molecule inhibitors of ST8SIA1, we developed a homolog of ST8SIA1 using the crystal structure of ST8SIA3 (PDB ID: 5B09) as a template. ST8SIA3 shares 32% identity and 54% similarity with this crystal structure. To this model we docked GM3, the substrate for the ST8SIA1 enzyme and analyzed their interactions. Next, we used this model to dock the compound library to identify potential inhibitors of ST8SIA1. To that end, we performed structure-based virtual screening or virtual ligand screening of 10,000 compounds based on the structure of GM3 (>70% identity), which we retrieved from the ChemSpider database. Similarly, we retrieved 1500 compounds based on the parent structure of flavone (>70% identity) from the ZINC database. After further filtration and selection of these structures, we ranked these compounds on the basis of 11 different scoring functions in DS 4.5. We selected the top candidate (ZINC02886919) for further functional characterization. ZINC02886919 had a predicted binding affinity (-logKd) of 6.17, with which we calculated the predicted Kd as 0.812 μM. Visualization analysis demonstrated that ZINC02886919 is involved in both polar and hydrophobic interactions in the binding pocket. We then synthesized ZINC02886919 and pursued pharmacological and in vitro assays. Treatment of SUM159 cells with ZINC02886919 decreased the percentage of GD2+ BCSCs by 5- to 10-fold within 72h in a concentration-dependent manner (from 31% to 6%). In addition, ZINC02886919 inhibited soft agar colony and mammosphere formation for SUM159 cells by 10- to 20-fold. These data indicated that the ST8SIA1 homolog model that we developed shares structural similarity with native human ST8SIA1 protein and that ZINC02886919 is a potential inhibitor of ST8SIA1 activity and thus BCSC function. In conclusion, inhibition of ST8SIA1 expression or activity inhibits BCSC function, TNBC tumor growth, and metastasis. The ST8SIA1 inhibitor ZINC02886919 inhibits GD2 expression and mammosphere and soft agar colony formation in vitro. The effect of ZINC02886919 on tumor growth and metastasis is currently being tested in vivo. Citation Format: Appalaraju Jappupilli, Khoa Nguyen, Stanley Ly, Michael Andreeff, Prashen Chelikani, Venkata Lokesh Battula. Characterization of a small molecule inhibitor for GD3 synthase (ST8SIA1), a novel target in breast cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1169.

Positron Emission Tomography Detects In Vivo Expression of Disialoganglioside GD2 in Mouse Models of Primary and Metastatic Osteosarcoma.

The cell membrane glycolipid GD2 is expressed by multiple solid tumors, including 88% of osteosarcomas and 98% of neuroblastomas. However, osteosarcomas are highly heterogeneous, with many tumors exhibiting GD2 expression on <50% of the individual cells, while some tumors are essentially GD2-negative. Anti-GD2 immunotherapy is the current standard of care for high-risk neuroblastoma, but its application to recurrent osteosarcomas, for which no effective therapies exist, has been extremely limited. This is, in part, because the standard assays to measure GD2 expression in these heterogeneous tumors are not quantitative and are subject to tissue availability and sampling bias. To address these limitations, we evaluated a novel, sensitive radiotracer [64Cu]Cu-Bn-NOTA-hu14.18K322A to detect GD2 expression in osteosarcomas (six patient-derived xenografts and one cell line) in vivo using positron emission tomography (PET). Tumor uptake of the radiolabeled, humanized anti-GD2 antibody [64Cu]Cu-Bn-NOTA-hu14.18K322A was 7-fold higher in modestly GD2-expressing osteosarcomas (32% GD2-positive cells) than in a GD2-negative tumor (9.8% vs. 1.3% of the injected dose per cc, respectively). This radiotracer also identified lesions as small as 29 mm3 in a 34% GD2-positive model of metastatic osteosarcoma of the lung. Radiolabeled antibody accumulation in patient-derived xenografts correlated with GD2 expression as measured by flow cytometry (Pearson r = 0.88, P = 0.01), distinguishing moderately GD2-expressing osteosarcomas (32%-69% GD2-positive cells) from high GD2 expressors (>99%, P < 0.05). These results support the utility of GD2 imaging with PET to measure GD2 expression in osteosarcoma and thus maximize the clinical impact of anti-GD2 immunotherapy. SIGNIFICANCE: In situ assessment of all GD2-positive osteosarcoma sites with a novel PET radiotracer could significantly impact anti-GD2 immunotherapy patient selection and enable noninvasive probing of correlations between target expression and therapeutic response.

Human Sialic acid O-acetyl esterase (SIAE) \u2013 mediated changes in sensitivity to etoposide in a medulloblastoma cell line

Medulloblastoma (MB), the most common malignant paediatric brain tumour occurs in the cerebellum. Advances in molecular genomics have led to the identification of defined subgroups which are associated with distinct clinical prognoses. Despite this classification, standard therapies for all subgroups often leave children with life-long neurological deficits. New therapeutic approaches are therefore urgently needed to reduce current treatment toxicity and increase survival for patients. GD3 is a well-studied ganglioside which is known to have roles in the development of the cerebellum. Post-partum GD3 is not highly expressed in the brain. In some cancers however GD3 is highly expressed. In MB cells GD3 is largely acetylated to GD3A. GD3 is pro-apoptotic but GD3A can protect cells from apoptosis. Presence of these gangliosides has previously been shown to correlate with resistance to chemotherapy. Here we show that the GD3 acetylation pathway is dysregulated in MB and as a proof-of-principle we show that increased GD3 expression sensitises an MB cell line to etoposide.

High GD2 expression defines breast cancer cells with enhanced invasiveness

IntroductionBreast cancer is the most frequently diagnosed cancer among women. Cancer stem cells (CSCs) are suggested to be responsible for tumor initiation, progression, metastasis, recurrence and drug resistance. This study was conducted to evaluate the clinical significance of GD2, a newly suggested CSC marker and two other traditional CSC markers, CD44 and CD24 in breast cancer patients. Material and methodsA total of 168 primary breast cancer tissues were evaluated in terms of GD2, CD44 and CD24 expression using tissue microarray. Then, the correlation of expression levels of these markers with patients' clinicopathological characteristics was assessed. ResultsHigher GD2 expression was mainly found in patients with advanced histological grade (p = 0.02), presence of lymph node invasion (p = 0.04), larger size of tumors (p = 0.04) and older age (p = 0.04). Breast cancer samples with advanced histological grade also showed higher CD44 (p = 0.03) and CD24 expression (p = 0.05). A significant positive association was found between increased CD24 expression and lymph node involvement (p = 0.01). Furthermore, GD2-high/CD44-high/CD24-low phenotype was frequently seen in breast cancer samples with positive lymph node involvement (p = 0.05). ConclusionIn summary, increased expression of GD2 may define more aggressive tumor behavior in breast cancer. GD2 can well be considered as a diagnostic and prognostic marker in breast cancer.

Therapeutic targeting of the immunosuppressive axis in ovarian cancer using dietary supplements

Abstract Ovarian cancer is the deadliest gynecologic malignancy worldwide and mortality rates have remained steady despite advances in treatment. A major obstacle for current therapies is overcoming tumor immunosuppression, therefore targeting inhibitory factors produced in the tumor microenvironment could enhance antitumor responses. Natural Killer T (NKT) cells, a subset of T cells that recognize glycolipid antigens presented by CD1d, are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which binds with high affinity to CD1d, thereby suppressing NKT cell activation. In addition to GD3, we have identified vascular endothelial growth factor (VEGF) as another immunosuppressive factor secreted by ovarian cancers. We found that VEGF inhibition results in decreased GD3 expression in human ovarian cancer cell lines and restoration of NKT cell responses. In addition, we found that treatment with VEGF can directly impair NKT cell functional responses. To therapeutically target the VEGF-GD3 signaling cascade, mouse and human ovarian cancer cell lines were treated with triptolide, a phytochemical which has demonstrated potent antitumor activity in vitro and in vivo. Treatment with triptolide reduces VEGF secretion by ovarian cancer cell lines and results in a concomitant decrease in the expression of GD3 synthase. In addition, pre-treatment of ovarian cancer cells with triptolide improves the ability of NKT cells to recognize and lyse the cancer cells. Thus, we have identified a novel therapeutic strategy that can be used to block the production of immunosuppressive factors, thereby restoring NKT cell-mediated antitumor immune responses in ovarian cancer.

Frequency of ALK and GD2 Expression in Neuroblastoma

Background: The aim of this study was to elucidate the significance of immunohistochemical staining patterns of ALK and GD2 in peripheral neuroblastic tumors with different stages and favorable/unfavorable features. Materials and methods: 32 neuroblastomas, 7 ganglioneuroblastomas, and 1 ganglioneuroma cases were immunohistochemically stained with ALK and GD2, and the expressions were graded and correlated with differentiation, size, and favorable/unfavorable histology. Results: There was no statistically significant correlation between ALK immunopositivity and tumor differentiation or stage. Although there was no statistically significant correlation between GD2 immunopositivity and stage, the intensity and prevalence of GD2 immunostaining were statistically significantly higher in the well differentiated group and in tumors which were smaller than 10 cm. Conclusion: GD2 immunostaining levels correlated with tumor differentiation and size. ALK immunostaining was not related to tumor differentiation or stage.

Combined sialic acid and histone deacetylase (HDAC) inhibitor treatment up-regulates the neuroblastoma antigen GD2

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2-targeted immunotherapy. Here, we report that the cellular sialylation pathway, as well as epigenetic reprogramming, strongly modulates GD2 expression in human and mouse neuroblastoma cell lines. Recognition of GD2 by the 14G2a antibody is sialic acid-dependent and was blocked with the fluorinated sialic acid mimetic Ac53FaxNeu5Ac. Interestingly, sialic acid supplementation using a cell-permeable sialic acid analogue (Ac5Neu5Ac) boosted GD2 expression without or with minor alterations in overall cell surface sialylation. Furthermore, sialic acid supplementation with Ac5Neu5Ac combined with various histone deacetylase (HDAC) inhibitors, including vorinostat, enhanced GD2 expression in neuroblastoma cells beyond their individual effects. Mechanistic studies revealed that Ac5Neu5Ac supplementation increased intracellular CMP-Neu5Ac concentrations, thereby providing higher substrate levels for sialyltransferases. Furthermore, HDAC inhibitor treatment increased mRNA expression of the sialyltransferases GM3 synthase (ST3GAL5) and GD3 synthase (ST8SIA1), both of which are involved in GD2 biosynthesis. Our findings reveal that sialic acid analogues and HDAC inhibitors enhance GD2 expression and could potentially be employed to boost anti-GD2 targeted immunotherapy in neuroblastoma patients.

EZH2 Inhibition in Ewing Sarcoma Upregulates GD2 Expression for Targeting with Gene-Modified T Cells.

Chimeric antigen receptor (CAR) engineering of Tcells allows one to specifically target tumor cells via cell surface antigens. A candidate target in Ewing sarcoma is the ganglioside GD2, but heterogeneic expression limits its value. Here we report that pharmacological inhibition of Enhancer of Zeste Homolog 2 (EZH2) at doses reducing H3K27 trimethylation, but not cell viability, selectively and reversibly induces GD2 surface expression in Ewing sarcoma cells. EZH2 in Ewing sarcoma cells directly binds to the promoter regions of genes encoding for two key enzymes of GD2 biosynthesis, and EZH2 inhibition enhances expression of these genes. GD2 surface expression in Ewing sarcoma cells is not associated with distinct invitro proliferation, colony formation, chemosensitivity, or invivo tumorigenicity. Moreover, disruption of GD2 synthesis by gene editing does not affect its invitro behavior. EZH2 inhibitor treatment sensitizes Ewing sarcoma cells to effective cytolysis by GD2-specific CAR gene-modified Tcells. In conclusion, we report a clinically applicable pharmacological approach for enhancing efficacy of adoptively transferred GD2-redirected Tcells against Ewing sarcoma, by enabling recognition of tumor cells with low or negative target expression.

Abstract A131: Targeting B7-H3 (CD276) in neuroblastoma: In vitro evaluation of Fc-optimized antibodies and immunocytokines

Abstract Introduction: Targeting disialoganglioside GD2 with monoclonal antibodies (mAbs) significantly improves survival in high-risk neuroblastoma (NB) patients after autologous or allogeneic SCT. However, GD2 expression is heterogeneous and the recently approved anti-GD2 mAb CH14.18 causes severe adverse effects, e.g., neuropathy and neuropathic pain due to neurotoxicity. Additional or alternative target antigens might improve therapy. B7-H3 belongs to the B7-CD28 family and is thought to function as an immune checkpoint by regulating T and NK cell response. B7-H3 is highly overexpressed on many solid tumors, correlating with poor prognosis and outcome. However, on healthy tissue its protein expression is very limited, thus making B7-H3 an interesting target for cancer immunotherapy. Therefore, we evaluated the use of B7-H3 as an alternative to GD2 and investigated different anti-B7-H3 mAb constructs and mAb-cytokine fusions (immunocytokines) for their ability to elicit antibody-dependenT-cellular cytotoxicity (ADCC). Methods: Derived from a parent anti-B7-H3 clone (HEK5-1B3), five additional mAb constructs were engineered: (1) chimeric with human IL-2 fusion (cHEK5-IL2), (2) chimeric and Fc-optimized (SDIE) w/o fusion (cHEK5opt), (3) cHEK5opt fused with human IL-2 (cHEK5opt-IL2), (4) cHEK5opt fused with human IL-15 (cHEK5opt-IL15), and (5) cHEK5-IL2 produced in rat myeloma YB2/0 to create an optimized low-fucose version (cHEX5LF-IL2). All IL-2 fusions were to the C-terminus of the light chain. The abilities of all six anti-B7-H3 mAb constructs and the GD2-specific mAb CH14.18 to mediate ADCC were compared in vitro in cytotoxicity assays using calcein release assays and the RTCA xCELLigence system. TargeT-cells: NB cell lines expressing high levels of B7-H3 but variable levels of GD2 (LAN-1, Kelly, SH-SY5Y). Effector cells: Human expanded NK cells (eNKs), expanded γ/δ T-cells and patient PBMCs after allogeneic SCT. Results: Except the parent clone, all anti-B7-H3 mAb constructs were able to elicit ADCC. TargeT-cell lysis of LAN-1 (high expression of both GD2 and B7-H3) mediated by the optimized anti-B7-H3 immunocytokines was comparable or even better than that mediated by CH14.18 (effectors: eNKs; calculated after 36 hrs.; in ascending order): Targets + effectors w/o mAb (24 %), parent pHEK5 (29 %), cHEK5opt (44 %), cHEK5-IL2 (76 %), cHEK5opt-IL15 (85 %), CH14.18 (90 %) and cHEK5opt-IL2 (97 %). Recently, we produced the low-fucose immunocytokine cHEX5LF-IL2. In a direct comparison with all other mAb constructs the cHEX5LF-IL2 immunocytokine mediated best targeT-cell lysis against SH-SY5Y (effectors: eNKs; calculated after 48 hrs.; in ascending order): Targets + effectors w/o mAb (31 %), CH14.18 (42 %), parent pHEK5 (46 %), cHEK5-IL2 (80 %), cHEK5opt (85 %), cHEK5opt-IL2 (93 %), cHEK5opt-IL15 (96 %) and low-fucose cHEX5LF-IL2 (100 %). Using expanded γ/δ T-cells of healthy donors we were able to confirm cHEX5LF-IL2 to be the most effective anti-B7-H3 mAb. Interestingly, cHEK5-IL2 showed comparable targeT-cell lysis; however, lysis was only transient while cHEX5LF-IL2 mediated permanent target cell lysis. Using patient PBMCs after receiving allogeneic SCT target cell lysis mediated by cHEX5LF-IL2 and CH14.18 was comparable. Calculated specific lysis of LAN-1 (after 96 hrs.; in ascending order): Targets + effectors w/o mAb (22 %), parent pHEK5 (26 %), cHEK5opt-IL2 (40 %), cHEK5opt (49 %), cHEK5-IL2 (57 %), cHEK5opt-IL15 (70 %), low-fucose cHEX5LF-IL2 (78 %) and CH14.18 (81 %). Conclusion: B7-H3 has been demonstrated to be a suitable alternative target antigen in case GD2 expression is low or absent. Fc-optimized mAbs and mAb-cytokine fusions targeting B7-H3 might increase the efficacy of immunotherapy in GD2-negative tumors and in combinatory approaches. So far, the low-fucose immunocytokine cHEX5LF-IL2 seems to be the most promising anti-B7-H3 construct. Citation Format: Florian Heubach, Patrick Schlegel, Latifa Zekri, Timo Manz, Sabine Schleicher, Armin Rabsteyn, Gundram Jung, Hans-Jörg Bühring, Stephen D. Gillies, Rupert Handgretinger, Peter Lang. Targeting B7-H3 (CD276) in neuroblastoma: In vitro evaluation of Fc-optimized antibodies and immunocytokines [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A131.

Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) as main O-acetylated sialic acid species of GD2 in breast cancer cells.

Mainly restricted to the nervous system in healthy adults, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. Interestingly, O-acetylated forms of GD2, not expressed in human peripheral nerve fibers, are highly expressed in GD2+ tumor cells. Very little information is known regarding the expression of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Here, we analyzed the expression of GD2, GD3 and their O-acetylated forms O-acetyl-GD2 (OAcGD2) and O-acetyl-GD3 (OAcGD3) in BC cells. We used Hs 578T and SUM159PT cell lines, as well as cell clones over-expressing GD3 synthase derived from MDA-MB-231 and MCF-7. Using flow cytometry and immunocytochemistry/confocal microscopy, we report that BC cells express b-series gangliosides GD3 and GD2, as well as significant amounts of OAcGD2. However, OAcGD3 expression was not detected in these cells. O-acetylation of gangliosides isolated from BC cells was examined by LC-MS analysis of sialic acid DMB-derivatives. We report that the main acetylated form of sialic acid expressed in BC gangliosides is 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). These results highlight a close interrelationship between Neu5,9Ac2 and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.

ST8SIA1 Regulates Tumor Growth and Metastasis in TNBC by Activating the FAK-AKT-mTOR Signaling Pathway.

Breast cancer stem-like cells (BCSC) are implicated in cancer recurrence and metastasis of triple-negative breast cancer (TNBC). We have recently discovered that ganglioside GD2 expression defines BCSCs and that ST8SIA1 regulates GD2 expression and BCSC function. In this report, we show that ST8SIA1 is highly expressed in primary TNBC; its expression is positively correlated with the expression of several BCSC-associated genes such as BCL11A, FOXC1, CXCR4, PDGFRβ, SOX2, and mutations in p53. CRISPR knockout of ST8SIA1 completely inhibited BCSC functions, including in vitro tumorigenesis and mammosphere formation. Mechanistic studies discovered activation of the FAK-AKT-mTOR signaling pathway in GD2+ BCSCs, and its tight regulation by ST8SIA1. Finally, knockout of ST8SIA1 completely blocked in vivo tumor growth and metastasis by TNBC cells. In summary, these data demonstrate the mechanism by which ST8SIA1 regulates tumor growth and metastasis in TNBC and identifies it as a novel therapeutic target.

Carbohydrate Targets for CAR T Cells in Solid Childhood Cancers.

Application of the CAR targeting strategy in solid tumors is challenged by the need for adequate target antigens. As a consequence of their tissue origin, embryonal cancers can aberrantly express membrane-anchored gangliosides. These are carbohydrate molecules consisting of a glycosphingolipid linked to sialic acids residues. The best-known example is the abundant expression of ganglioside GD2 on the cell surface of neuroblastomas which derive from GD2-positive neuroectoderm. Gangliosides are involved in various cellular functions, including signal transduction, cell proliferation, differentiation, adhesion and cell death. In addition, transformation of human cells to cancer cells can be associated with distinct glycosylation profiles which provide advantages for tumor growth and dissemination and can serve as immune targets. Both gangliosides and aberrant glycosylation of proteins escape the direct molecular and proteomic screening strategies currently applied to identify further immune targets in cancers. Due to their highly restricted expression and their functional roles in the malignant behavior, they are attractive targets for immune engineering strategies. GD2-redirected CAR T cells have shown activity in clinical phase I/II trials in neuroblastoma and next-generation studies are ongoing. Further carbohydrate targets for CAR T cells in preclinical development are O-acetyl-GD2, NeuGc-GM3 (N-glycolyl GM3), GD3, SSEA-4, and oncofetal glycosylation variants. This review summarizes knowledge on the role and function of some membrane-expressed non-protein antigens, including gangliosides and abnormal protein glycosylation patterns, and discusses their potential to serve as a CAR targets in pediatric solid 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.