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BackgroundCD19-targeted chimeric antigen receptor therapies (CAR19) have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CAR19-treated patients experienced progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant number of virally engineered CAR-T cells.MethodsUsing a highly efficientpiggyBactransposon-based vector,Quantum pBac, we developed a virus-free cell engineering system,Quantum CART (qCART™), for development and production of multiplex CAR-T therapies.ResultsHere, we demonstratedin vitro and in vivothat consistent, robust, and functional CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using theqCART™ system for clinical application.qCART™-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug.ConclusionsTheqCART™ system is an elegant system for the manufacturing of CAR-T products having all the desired CAR-T therapy attributes. We believe that the simplicity of manufacturing multiplex CAR-T cells using theqCART™ system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future.What is already known on this topicDespite the considerable success which has been achieved with CD19-targeted chimeric antigen receptor therapies (CAR19), >50% of CAR19-treated patients still experienced progressive disease. Therefore, there is a need to further improve CAR19 therapies. Current CAR19 therapies commonly utilize virus-based cell engineering methods. CAR-T production using these methods face multiple hurdles, including difficulties to efficiently, cost-effectively, and consistently manufacture clinically relevant number of CAR-T cells. We have previously used a highly efficientpiggyBactransposon-based vector,Quantum pBac, to establishQuantum CART(qCART™) which is a virus-free cell engineering system for development and production of multiplex CAR-T therapies.What this study addsIn this report, we further demonstratein vitroandin vivothat consistent, robust, and functional iCasp9-regulatable, CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using theqCART™ system for clinical application. These cells possess all the desired attributes for ensuring therapeutic efficacy in CAR-T therapy, including high CAR-TSCM, balanced CD8/CD4 ratio, low exhaustion and senescence marker expressions, and highex vivoandin vivoexpansion capacity. Importantly, we show thatqCART™-manufactured CAR-T cells from hematological cancer patients expanded efficiently, effectively eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. We believe that the simplicity of manufacturing multiplex CAR-T cells using theqCART™ system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future.How this study might affect research, practice or policyOur findings demonstrate thatqCART™ is a virus-free CAR-T engineering system for manufacturing CAR-TSCMcells from either healthy donors or hematological cancer patients, that possess all the desired attributes for a successful CAR-T therapy. These cells expanded efficiently, rapidly eradicated tumors, and can be safely controlled via activation of iCasp9. We expect that this simple yet robust system for manufacturing multiplex CAR-T cells will advance the CAR-T field.

Background: Globo H (GH), a Globo-series glycosphingolipid (GSL), is highly expressed in epithelial tumors, such as colon, endometrial, gastric, pancreatic, lung, prostate, and breast cancers. Aberrant expression of Globo H has been reported to be associated with the metastatic potential and poor prognosis of these cancers. However, in normal tissues, GH expression is limited to the secretory borders of apical epithelial cells, making it difficult to access by the immune system. GH is therefore a promising target for anticancer therapeutics. Clinical studies with the Globo H vaccines (OBI-822 and OBI-833) and the humanized anti-Globo H antibody (OBI-888) demonstrating an excellent safety profile. In recent years, cell therapy using chimeric antigen receptor T (CAR T) cells have shown impressive clinical outcomes. As a result, development of CAR T targeting GH may offer a novel anticancer therapeutic agent. Aim: The aim of this study was to develop a CAR T cell therapy targeted against Globo H (obi-R007) using lentivirus-mediated genetic engineering with a proprietary Globo H-specific antibody. Methods: We conducted in vitro and in vivo studies to determine the characteristics of GH CAR T obi-R007. Results: Ex vivo expansion of obi-R007 from healthy donors ranged from 40- to 200-fold post-activation for 10 days. Previous studies have suggested that less differentiation and fewer exhaustion markers of CAR T cells are beneficial for therapeutic persistence. In our study, there was less differentiation in the CD62L+ population (TSCM and TCM) in greater than 80% of obi-R007 cells, and no significant exhaustion markers (PD-1, LAG-3, etc.) were detected. obi-R007 showed specific cytotoxicity against Globo H-positive tumor cells with an E/T ratio of 0.5:1~2:1 and exhibited Globo H-specific activation through the expression of CD69, CD25, and Granzyme B as well as the release of Interferon γ and IL-2. Moreover, the activation and proliferation of obi-R007 were dependent on the presence of target cells. All the in vitro results indicate the specific targeting of obi-R007 to Globo H resulting in cytotoxic T cell responses. In the in vivo study, the efficacy of obi-R007 was demonstrated by adoptive cell transfer in several xenograft models and obi-R007 was shown to be persistent under multiple tumor challenges in the NCI-N87 gastric cancer model. Luminance labelling of CAR T cells showed that the distribution of CAR T cells in mice was specific to the tumor site and lymphoid organs suggesting a homing activity of the CAR T cells. Furthermore, no obviously physiological toxicity was observed in vivo. Conclusion: In conclusion, our in vitro and in vivo pharmacology and preliminary toxicology studies support clinical development of Globo H CAR T immunotherapy for patients with cancer. Citation Format: Jiann-Shiun Lai, Shiou-Ling Jian, Woan-Eng Chan, Ming-Tain Lai. Globo H-targeted CAR T cell cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4084.

Immunotherapy aiming to rescue or boost antitumor immunity is an emerging strategy for treatment of cancers. The efficacy of immunotherapy is strongly controlled by the immunological milieu of cancer patients. Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cell populations with immunosuppressive functions accumulating in individuals during tumor progression. The signaling mechanisms of MDSC activation have been well studied. However, there is little known about the metabolic status of MDSCs and the physiological role of their metabolic reprogramming. In this study, we discovered that myeloid cells upregulated their glycolytic genes when encountered with tumor-derived factors. MDSCs exhibited higher glycolytic rate than their normal cell compartment did, which contributed to the accumulation of the MDSCs in tumor-bearing hosts. Upregulation of glycolysis prevented excess reactive oxygen species (ROS) production by MDSCs, which protected MDSCs from apoptosis. Most importantly, we identified the glycolytic metabolite, phosphoenolpyruvate (PEP), as a vital antioxidant agent able to prevent excess ROS production and therefore contributed to the survival of MDSCs. These findings suggest that glycolytic metabolites have important roles in the modulation of fitness of MDSCs and could be potential targets for anti-MDSC strategy. Targeting MDSCs with analogs of specific glycolytic metabolites, for example, 2-phosphoglycerate or PEP may diminish the accumulation of MDSCs and reverse the immunosuppressive milieu in tumor-bearing individuals.

Breast cancer is the most common cancer and the second leading cause of cancer death in women. Except for surgery, radiotherapy, chemotherapy and targeting therapy, breast cancer vaccine is now actively developed. However, the outcome of most therapeutic cancer vaccines was not satisfying probably due to the induction of immunosuppression in cancer patients. Myeloid-derived suppressor cells (MDSCs) are one of the major immunoregulatory cell populations, which play important roles not only in T-cell suppression but also in angiogenesis, lymphangiogenesis and metastasis during tumor progression. MDSCs are heterogeneous populations of immature myeloid cells, which expand and developed in responses to growth factors, cytokines and chemokines derived from cancer cells and then are released from bone marrow to peripheral tissues including tumor site. It is well known as tumor cells could influence the activation and proliferation of myeloid cells through release of cytokines during tumor progression to result in the accumulation of large number of MDSCs in cancer patients. However little is known about the relevance of metabolic status and accumulation of MDSCs in cancer patients during tumor progression. We therefore try to elucidate the metabolic changes during differentiation of myeloid cells into MDSCs. To intensively study the transcriptional program of MDSCs in comparison to their healthy counterparts, we perform DNA microarray analysis using mRNAs from monocytic MDSCs, granulocytic MDSCs from primary tumor sites of 4T1 tumor-bearing mice, monocytes and neutrophils from bone marrow of healthy mice. MDSCs up-regulated mRNA levels of genes related to angiogenesis and lymphangiogenesis e.g. VEGFa, angiopoietin 1, PROK2 (BV8), MMP9, MMP12, MMP13 and MMP14, genes of pro-inflammatory cytokines e.g. IL-1α, IL-6 and TNFα, genes of immunosuppressive molecules e.g. arginase 1, iNOS, PD-L1, PD-L2, CD83 and CD155. We are currently examining the metabolomics of these MDSCs and their healthy counterparts and expect to prove the association of certain metabolic changes with the differentiation of MDSCs. Citation Format: Li-Rung Huang, Shiou-Ling Jian, Yi-Jyun Jhou. The influence of breast cancer on the metabolic status of myeloid cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B78.