Ewing sarcoma (ES) is thought to arise from mesenchymal stem cells and is the second most common bone sarcoma in pediatric patients and young adults. Given the dismal overall outcomes and very intensive therapies used, there is an urgent need to explore and develop alternative treatment modalities including immunotherapies. In this article, we provide an overview of ES biology, features of ES tumor microenvironment (TME) and review various tumor-associated antigens that can be targeted with immune-based approaches including cancer vaccines, monoclonal antibodies, T cell receptor-transduced T cells, and chimeric antigen receptor T cells. We highlight key reasons for the limited efficacy of various immunotherapeutic approaches for the treatment of ES to date. These factors include absence of human leukocyte antigen class I molecules from the tumor tissue, lack of an ideal surface antigen, and immunosuppressive TME due to the presence of myeloid-derived suppressor cells, F2 fibrocytes, and M2-like macrophages. Lastly, we offer insights into strategies for novel therapeutics development in ES. These strategies include the development of gene-modified T cell receptor T cells against cancer–testis antigen such as XAGE-1, surface target discovery through detailed profiling of ES surface proteome, and combinatorial approaches. In summary, we provide state-of-the-art science in ES tumor immunology and immunotherapy, with rationale and recommendations for future therapeutics development.
Five reactions were rate-accelerated relative to the standard reflux workup in both multi-mode and mono-mode microwave ovens, and the results were compared to determine whether the sequential processing of a mono-mode unit could provide for better lab logistics and pedagogy. Conditions were optimized so that yields matched in both types of microwave ovens for a Diels−Alder cycloaddition, Wittig salt formation, Fischer esterifications, an E2 alkyne formation, and Williamson ether synthesis. Typically, a 10-fold rate acceleration was observed under mono-mode heating versus multi-mode heating, reducing the total run-time between 1.5 and 3.0 min per sample, which rivals the batch run-time of a multi-mode unit in ∼16 student lab sections. Thus, the mono-mode microwave oven required a similar quantity of total reaction time in the lab, allowing students to run their experiments individually with less wait-time, competition for chemicals, equipment, and instrumentation and to complete the experiments in the lab period.
Ewing sarcoma is an aggressive tumor type with an age peak in adolescence. Despite the use of dose-intensified chemotherapy as well as radiation and surgery for local control, patients with upfront metastatic disease or relapsed disease have a dismal prognosis, highlighting the need for additional therapeutic options. Different types of immunotherapies have been investigated with only very limited clinical success, which may be due to the presence of immunosuppressive factors in the tumor microenvironment. Here we provide an overview on different factors contributing to Ewing sarcoma immune escape. We demonstrate ways to target these factors in order to make current and future immunotherapies more effective and achieve deeper and more durable responses in patients with Ewing sarcoma.
Downregulation of HLA is one of the most common tumor escape mechanisms by enabling tumors to persist in the presence of tumor-reactive T cells. HLA loss is particularly common in children with high-risk neuroblastoma, who have a 50% long-term survival despite dose-intensive regimens. We have now developed an approach for the targeted induction of HLA to restore sensitivity of neuroblastoma cells to T cell-mediated killing. Using synNotch technology, we have generated T cells that, upon binding of the neuroblastoma surface antigen GD2, secrete IFN-γ without conferring direct cytotoxic activity (snGD2i). Treatment with snGD2i cells induces high and durable expression of HLA on neuroblastoma cells in vitro and in vivo and restores sensitivity to TCR-transgenic T cells targeting neuroblastoma-specific antigens. In contrast, treatment does not lead to upregulation of immune checkpoints or systemically increased levels of IFN-γ. Targeted delivery of IFN-γ using snGD2i cells is a promising new strategy to address immune escape in neuroblastoma.
B cell lymphoma is the most common hematologic malignancy in the United States. Although treatment options have greatly improved in the past several decades, outcomes for patients with relapsed B cell lymphoma remain poor. Chimeric antigen receptor (CAR) T cells have recently entered the clinic with promise to address the gap in effective therapies for patients relapsed B cell lymphoma. However, antigen loss and poor CAR T cell persistence has been shown to drive resistance to the widely approved CD19-targeted CAR in some patients, demonstrating the need for additional therapies. Here, we demonstrate CD229-targeted CAR T cell therapy as a promising option for the treatment of relapsed B cell lymphoma, addressing an important group of patients with typically poor outcomes. CD229 is an immune-modulating receptor expressed on the surface of B cells that we recently found to be highly expressed in the plasma cell neoplasm multiple myeloma (Radhakrishnan et al. 2020). We utilized semi-quantitative PCR and flow cytometry to assess whether CD229 is also expressed on malignant B cells earlier in development as found in B cell lymphoma. Expression analysis revealed the presence of CD229 in a panel of 11 B cell lymphoma cell lines and 45 primary B cell lymphoma samples comprising several subsets of disease including aggressive B cell lymphomas such as diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL) and Burkitt lymphoma as well as indolent subtypes of B cell lymphoma including chronic lymphoblastic leukemia (CLL) and follicular lymphoma. Of note, CD229 was found to be overexpressed on primary B cell lymphoma cells when compared to autologous normal B cells. Given the high levels of CD229 expression throughout all B cell lymphoma subtypes analyzed, we generated CD229 CAR T cells in order to determine whether CAR T cell therapy is an effective way to target CD229 expressing B cell lymphoma cells. CD229 CAR T cells exhibited robust cytotoxicity when cocultured with B cell lymphoma cell lines and primary samples characterized by significant production of TH1 cytokines IL-2, TNF and IFNγ and rapid loss of B cell lymphoma cell viability when compared to control CAR T cells lacking an antigen binding scFv domain (∆scFv CAR T cells). In vivo analysis revealed effective tumor control in NSG mice carrying B cell lymphoma cell lines JeKo-1 (MCL) and DB (DLBCL) when treated with CD229 CAR T cells versus ∆scFv CAR T cells. Finally, we sought to determine the efficacy of CD229 CAR T cells in the context of CD19 CAR T cell therapy relapse. Here, a 71-year-old patient with CLL had an initial response when treated with CD19 CAR T cells but quickly relapsed only 2 months after treatment. Malignant cells from the CLL patient retained CD229 expression as identified by flow cytometry and an ex vivo coculture with CD229 CAR T cells revealed robust killing of CLL cells by CD229 CAR T cells. Transfer of antigen from target cell to CAR T cell by trogocytosis was recently suggested to drive relapse following CAR T cell therapy by decreasing antigen on tumor cells and promoting CAR T cell fratricide (Hamieh et al. 2019). We cocultured CD19 and CD229 CAR T cells with primary CLL cells and assessed CD19 and CD229 expression as well as CAR T cell viability by flow cytometry. In contrast with CD19 CAR T cells, CD229 CARs did not strip their target antigen from the surface of CLL cells. The transfer of CD19 from CLL cells to CD19 CAR T cells resulted in poor CAR T cell viability while CD229 CAR T cell viability remained high following coculture. In summary, we demonstrate that CD229 is a promising therapeutic target in B cell lymphoma due to its high levels of expression throughout many subtypes of disease. CD229 CAR T cells effectively kill B cell lymphoma cells in vitro and control growth of aggressive B cell lymphomas in vivo. Finally, CD229 CAR T cells are effective against primary CLL cells from patients that have relapsed from CD19 CAR T cell therapy and do no exhibit antigen loss by trogocytosis. Taken together, these data suggest that CD229 CAR T cell therapy may be a promising option to address the poor outcomes for patients with relapsed B cell lymphoma. Disclosures No relevant conflicts of interest to declare.
Introduction: Patients with Down syndrome (DS) have an increased risk of hematological disorders, including transient abnormal myelopoiesis (TAM), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Twenty percent of patients with TAM subsequently develop myeloid neoplasm in the first 4 years of life. MDS represents a clonal aberration thought to be a pre-leukemic condition characterized clinically by cytopenias and erythroid, myeloid and/or megakaryocytic dysplasia in the bone marrow with or without increase in blasts and harbors a concordant, clone-specific mutation of GATA1. WHO 2016 classification of hematopoietic neoplasms does not distinguish between MDS and AML, as their overall prognosis appears to be similar. However, due to the rarity of this disorder, limited clinical and laboratory data is available, contributing to difficulties in establishing the diagnosis. Here we describe our center's recent experience with the diagnosis and molecular findings of myeloid neoplasm associated with Down syndrome (MN-DS). Design/Method: Retrospective review of the patient's electronic medical record and review of the literature was conducted. Routine karyotype, fluorescent in-situ hybridization (FISH) and next generation sequencing (NGS) studies were reviewed where available. Results: Six patients with DS diagnosed with AML or MDS were identified over a 3-year period. Mean age of the cohort was 18.5 (range 12-24) months with a slight female predominance. Three patients had a history of TAM, all of which resolved without intervention. Three patients had asymptomatic thrombocytopenia after birth without blasts or GATA1 mutation confirmation. One of the three patients with a history of TAM presented with overt AML, while in the others diagnosis was challenging. By WHO 2008 classification of myeloid neoplasms, four patients had refractory anemia with excess blasts, one had refractory cytopenia with multilineage dysplasia, and one had AML. For two patients, in whom myeloid directed next generation sequencing was obtained, mutations were found in GATA1, EZH2, and NRAS. One of the patients in our series presented with AML with gain of MECOM, RPN1 loss and D5S23 deletion by FISH and succumbed to relapsed disease. All patients were treated per Children's Oncology Group AAML1531 arm A protocol that included 3 induction cycles and 2 intensification cycles, except for a single patient that received one cycle per AAML0431 and completed therapy per AAML1531 arm B high risk due to persistent disease following initial induction cycle. Two patients are currently receiving treatment, three have no evidence of disease recurrence on follow up ranging from 2 to 18 months, and one of the patients has died due to relapsed/refractory disease. Conclusions: We present six cases of MN-DS in patients less than four years of age. Our cohort is representative of the diversity encountered in this rare disease including patients with 1) isolated cytopenia in the absence of overt morphological findings, 2) myelodysplasia, and 3) AML. In our patient with overt AML there were karyotypic features such as gain of MECOM, which with specific translocation partners has previously been described to portend a poor prognosis. This and other cytogenetic features perhaps warrant further investigation given our patient's refractory disease. In the patient with refractory cytopenia without blasts, there was a subpopulation of cells identified by NGS panel showing mutations in GATA1, EZH2, and NRAS that led to a diagnosis of MDS/MN-DS. Four of the patients had aberrant myeloid populations and dysplasia fitting diagnostic criteria for MDS. Establishing the clonal nature of the disease either by karyotype/FISH or NGS may help with the identification, treatment and prognostication of this unique patient population, and may aid in the diagnosis of MN-DS, which may be challenging in patients with DS once they have recovered from TAM. Disclosures No relevant conflicts of interest to declare.
Chimeric Antigen Receptor (CAR) T cells have shown remarkable anti-tumor activity in various B cell malignancies. However, expanding their use to solid tumors has proven difficult due to the lack of sufficiently tumor-specific target antigens. Restricting CAR T cells from trafficking to certain healthy tissues showing shared expression of an otherwise tumor-specific target antigen may open up new avenues to the treatment of solid tumors with CAR T cells. We developed an electroporation-based CRISPR/Cas9 approach targeting integrin a4 (ITGA4), a key component of the T cell adhesion heterodimer VLA-4 required for blood-brain-barrier migration via the endothelial adhesion protein VCAM-1. We characterize ITGA4 knockout kinetics in human T cells by flow cytometry, determine its effect on T cell adhesion to VCAM-1 using a custom in vitro adhesion assay, determine tissue distribution of ITGA4ko cells in a mouse model and characterize the anti-tumor activity of ITGA4ko CAR T cells using a luminescence-based cytotoxicity assay. Near-complete ITGA4 knockout could be achieved during a standard 12-day CAR T cell production process and significantly decreased adhesion of human T cells to VCAM-1. ITGA4ko T cells appeared to be efficiently redirected from the brain to the bone marrow which was validated by flow cytometry showing almost a complete lack of T cells in the brain after ITGA4 knockout. Importantly, knockout did not significantly change CAR T cell expansion or anti-tumor activity. In conclusion, we show that ITGA4 knockout is feasible, results in efficient exclusion of T cells from the brain without affecting CAR T cell function, representing a promising approach to shape CAR T cell selectivity for the treatment of solid tumors.
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