bims-catcri Biomed News
on CAR-T cells, CRISPR and cancer
Issue of 2022–08–28
eleven papers selected by
Lisa Dwane, AstraZeneca
  1. DLK1-directed chimeric antigen receptor T-cell therapy for hepatocellular carcinoma.
  2. Chimeric Antigen Receptor T Cells Targeting Cell Surface GRP78 to Eradicate Acute Myeloid Leukemia.
  3. Road testing new CAR design strategies in multiple myeloma.
  4. Overcoming tumor resistance mechanisms in CAR-NK cell therapy.
  5. Epigenetic engineering for optimal CAR-T cell therapy.
  6. Rationale and Clinical Research Progress on PD-1/PD-L1-Based Immunotherapy for Metastatic Triple-Negative Breast Cancer.
  7. Modern Immunotherapy in the Treatment of Triple-Negative Breast Cancer.
  8. CAR T cells targeting the ganglioside NGcGM3 control ovarian tumors in the absence of toxicity against healthy tissues.
  9. RASA2 ablation in T cells boosts antigen sensitivity and long-term function.
  10. scRNA-seq of gastric tumor shows complex intercellular interaction with an alternative T cell exhaustion trajectory.
  11. Emerging advances in engineered macrophages for tumor immunotherapy.
  1. Liver Int. 2022 Aug 24.
    DLK1-directed chimeric antigen receptor T-cell therapy for hepatocellular carcinoma.
    Yangyang Zhai, Kunyan He, Liyu Huang, Xuyang Shang, Guangxing Wang, Guandou Yuan, Ze-Guang Han.
       BACKGROUND: Delta-like homolog 1 (DLK1), a transmembrane protein, is highly expressed in hepatocellular carcinoma (HCC). We explored whether DLK1-directed chimeric antigen receptor (CAR) T cells can specifically eliminate DLK1-positive HCC cells and serve as a therapeutic strategy for HCC immunotherapy.
    METHODS: We first characterized a homemade anti-human DLK1 monoclonal antibody, sequenced the single-chain Fragment variable (scFv) and integrated it into the second-generation CAR lentiviral vector, and then developed the DLK1-directed CAR-T cells. The cytotoxic activities of DLK1-directed CAR-T cells against different HCC cells were evaluated in vitro and in vivo.
    RESULTS: The genetically modified human T cells with the DLK1-directed CARs produced cytotoxic activity against DLK1-positive HCC cells. Additionally, the DLK1-directed CARs enhanced T cell proliferation and activation in a DLK1-dependent manner. Interestingly, the DLK1-targeted CAR-T cells significantly inhibited both subcutaneous and peritoneal xenograft tumors derived from human liver cancer cell lines HepG2 or Huh-7.
    CONCLUSION: DLK1-directed CAR T cells specifically suppresses DLK1-positive HCC cells in vitro and in vivo. This study provides a novel transmembrane antigen DLK1 as a potential therapeutic target appropriate for CAR-T cell therapy, which may be further developed as a clinical therapeutic strategy for HCC immunotherapy.
    Keywords:  CAR-T; DLK1; HCC; immunotherapy
    DOI:  https://doi.org/10.1111/liv.15411
  2. Front Cell Dev Biol. 2022 ;10 928140
    Chimeric Antigen Receptor T Cells Targeting Cell Surface GRP78 to Eradicate Acute Myeloid Leukemia.
    Wei Yu, Hang Zhang, Yuncang Yuan, Jie Tang, Xinchuan Chen, Ting Liu, Xudong Zhao.
      Acute myeloid leukemia (AML) is a serious, life-threatening hematological malignancy. The treatment outcome of relapsed or refractory AML patients remains dismal, and new treatment options are needed. Chimeric antigen receptor (CAR) T cells have been successful in improving the prognosis for B-lineage acute lymphoblastic leukemia and lymphoma by targeting CD19. However, CAR T-cell therapy for AML is still elusive, owing to the lack of a tumor-specific cell surface antigen and spare hematopoietic stem cells (HSCs). This study generated a novel CAR construction that targets the cell surface protein glucose-regulated protein 78 (GRP78) (csGRP78). We confirmed that GRP78-CAR T cells demonstrate an anti-tumor effect against human AML cells in vitro. In xenograft models, GRP78-CAR T cells effectively eliminate AML cells and protect mice against systemic leukemia, in the meanwhile, prolonging survival. In addition, GRP78-CAR T cells also specifically eradicate the primary AML patient-derived blast. In particular, GRP78-CAR T cells spare normal HSCs, highlighting that GRP78-CAR is a promising approach for the therapy of AML.
    Keywords:  CAR T cell; GRP78; acute myeloid leukemia (AML); cell surface; hematopoietic stem cells (HSCS)
    DOI:  https://doi.org/10.3389/fcell.2022.928140
  3. Front Immunol. 2022 ;13 957157
    Road testing new CAR design strategies in multiple myeloma.
    Priyanka S Rana, Elena V Murphy, Jeries Kort, James J Driscoll.
      A deeper understanding of basic immunology principles and advances in bioengineering have accelerated the mass production of genetically-reprogrammed T-cells as living drugs to treat human diseases. Autologous and allogeneic cytotoxic T-cells have been weaponized to brandish MHC-independent chimeric antigen receptors (CAR) that specifically engage antigenic regions on tumor cells. Two distinct CAR-based therapeutics designed to target BCMA are now FDA-approved based upon robust, sustained responses in heavily-pretreated multiple myeloma (MM) patients enrolled on the KarMMa and CARTITUDE-1 studies. While promising, CAR T-cells present unique challenges such as antigen escape and T-cell exhaustion. Here, we review novel strategies to design CARs that overcome current limitations. Co-stimulatory signaling regions were added to second-generation CARs to promote IL-2 synthesis, activate T-cells and preclude apoptosis. Third-generation CARs are composed of multiple co-stimulatory signaling units, e.g., CD28, OX40, 4-1BB, to reduce exhaustion. Typically, CAR T-cells incorporate a potent constitutive promoter that maximizes long-term CAR expression but extended CAR activation may also promote T-cell exhaustion. Hypoxia-inducible elements can be incorporated to conditionally drive CAR expression and selectively target MM cells within bone marrow. CAR T-cell survival and activity is further realized by blocking intrinsic regulators of T-cell inactivation. T-Cells Redirected for Universal Cytokine Killing (TRUCKs) bind a specific tumor antigen and produce cytokines to recruit endogenous immune cells. Suicide genes have been engineered into CAR T-cells given the potential for long-term on-target, off-tumor effects. Universal allo-CAR T-cells represent an off-the-shelf source, while logic-gated CAR T-cells are designed to recognize tumor-specific features coupled with Boolean-generated binary gates that then dictate cell-fate decisions. Future generations of CARs should further revitalize immune responses, enhance tumor specificity and reimagine strategies to treat myeloma and other cancers.
    Keywords:  CAR T-cell therapy; armored CAR; hypoxia; logic-gates; multiple myeloma; self-driving CAR
    DOI:  https://doi.org/10.3389/fimmu.2022.957157
  4. Front Immunol. 2022 ;13 953849
    Overcoming tumor resistance mechanisms in CAR-NK cell therapy.
    Antonio Valeri, Almudena García-Ortiz, Eva Castellano, Laura Córdoba, Elena Maroto-Martín, Jessica Encinas, Alejandra Leivas, Paula Río, Joaquín Martínez-López.
      Despite the impressive results of autologous CAR-T cell therapy in refractory B lymphoproliferative diseases, CAR-NK immunotherapy emerges as a safer, faster, and cost-effective approach with no signs of severe toxicities as described for CAR-T cells. Permanently scrutinized for its efficacy, recent promising data in CAR-NK clinical trials point out the achievement of deep, high-quality responses, thus confirming its potential clinical use. Although CAR-NK cell therapy is not significantly affected by the loss or downregulation of its CAR tumor target, as in the case of CAR-T cell, a plethora of common additional tumor intrinsic or extrinsic mechanisms that could also disable NK cell function have been described. Therefore, considering lessons learned from CAR-T cell therapy, the emergence of CAR-NK cell therapy resistance can also be envisioned. In this review we highlight the processes that could be involved in its development, focusing on cytokine addiction and potential fratricide during manufacturing, poor tumor trafficking, exhaustion within the tumor microenvironment (TME), and NK cell short in vivo persistence on account of the limited expansion, replicative senescence, and rejection by patient's immune system after lymphodepletion recovery. Finally, we outline new actively explored alternatives to overcome these resistance mechanisms, with a special emphasis on CRISPR/Cas9 mediated genetic engineering approaches, a promising platform to optimize CAR-NK cell function to eradicate refractory cancers.
    Keywords:  CAR NK cells; CAR persistence; CRISPR/Cas9; chimeric antigen receptor (CAR); genome editing; hematologic tumor; tumor microenvironment; tumor resistance
    DOI:  https://doi.org/10.3389/fimmu.2022.953849
  5. Cancer Sci. 2022 Aug 24.
    Epigenetic engineering for optimal CAR-T cell therapy.
    Yusuke Ito, Yuki Kagoya.
      Recent advancements in cancer immunotherapy, such as chimeric antigen receptor (CAR)-engineered T cell therapy and immune checkpoint therapy (ICT), have significantly improved the clinical outcomes of patients with several types of cancer. To broaden its applicability further and induce durable therapeutic efficacy, it is imperative to understand how antitumor T cells elicit cytotoxic functions, survive as memory T cells, or are impaired in their effector functions (exhausted) at the molecular level. T cell properties are regulated by their gene expression profiles, which are further controlled by epigenetic architectures, such as DNA methylation and histone modifications. Multiple studies have elucidated specific epigenetic genes associated with T-cell phenotypic changes. Conversely, exogenous modification of these key epigenetic factors can significantly alter T cell functions by extensively altering the transcription network, which can be applied in cancer immunotherapy by improving T cell persistence or augmenting effector functions. Since CAR-T cell therapy involves a genetic engineering step during the preparation of the infusion products, it would be a feasible strategy to additionally modulate specific epigenetic genes in CAR-T cells to improve their quality. Here, we review recent studies investigating how individual epigenetic factors play a crucial role in T-cell biology. We further discuss future directions to integrate these findings for optimal cancer immunotherapy.
    Keywords:  CAR-T cells; epigenetics; exhaustion; memory T cell; terminal differentiation
    DOI:  https://doi.org/10.1111/cas.15541
  6. Int J Mol Sci. 2022 Aug 10. pii: 8878. [Epub ahead of print]23(16):
    Rationale and Clinical Research Progress on PD-1/PD-L1-Based Immunotherapy for Metastatic Triple-Negative Breast Cancer.
    Yifan Ren, Jialong Song, Xinyi Li, Na Luo.
      Metastatic triple-negative breast cancer (mTNBC), a highly aggressive and malignant tumor, currently lacks an effective treatment. There has been some progress in the treatment of mTNBC with programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) immunotherapy in recent years. The combination of PD-1/PD-L1 inhibitors with other therapies is a noteworthy treatment strategy. Immunotherapy in combination with chemotherapy or small-molecule inhibitors still faces many challenges. Additionally, there are some new immunotherapy targets in development. We aimed to further evaluate the effectiveness and usefulness of immunotherapy for treating mTNBC and to propose new immunotherapy strategies. This review explains the rationale and results of existing clinical trials evaluating PD-1/PD-L1 inhibitors alone or in combination for the treatment of mTNBC. For patients with aggressive tumors and poor health, PD-1/PD-L1 inhibitors, either alone or in combination with other modalities, have proven to be effective. However, more research is needed to explore more effective immunotherapy regimens that will lead to new breakthroughs in the treatment of mTNBC.
    Keywords:  PD-1/PD-L1; anticancer drugs; cancer therapy; clinical trails; immune checkpoint blockade therapy; immunotherapy; metastatic triple-negative breast cancer
    DOI:  https://doi.org/10.3390/ijms23168878
  7. Cancers (Basel). 2022 Aug 10. pii: 3860. [Epub ahead of print]14(16):
    Modern Immunotherapy in the Treatment of Triple-Negative Breast Cancer.
    Jakub Wesolowski, Anna Tankiewicz-Kwedlo, Dariusz Pawlak.
      Triple-Negative Breast Cancer is a subtype of breast cancer characterized by the lack of expression of estrogen receptors, progesterone receptors, as well as human epidermal growth factor receptor 2. This cancer accounts for 15-20% of all breast cancers and is especially common in patients under 40 years of age, as well as with the occurring BRCA1 mutation. Its poor prognosis is reflected in the statistical life expectancy of 8-15 months after diagnosis of metastatic TNBC. So far, the lack of targeted therapy has narrowed therapeutic possibilities to classic chemotherapy. The idea behind the use of humanized monoclonal antibodies, as inhibitors of immunosuppressive checkpoints used by the tumor to escape from immune system control, is to reduce immunotolerance and direct an intensified anti-tumor immune response. An abundance of recent studies has provided numerous pieces of evidence about the safety and clinical benefits of immunotherapy using humanized monoclonal antibodies in the fight against many types of cancer, including TNBC. In particular, phase three clinical trials, such as the IMpassion 130, the KEYNOTE-355 and the KEYNOTE-522 resulted in the approval of immunotherapeutic agents, such as atezolizumab and pembrolizumab by the US Food and Drug Administration in TNBC therapy. This review aims to present the huge potential of immunotherapy using monoclonal antibodies directed against immunosuppressive checkpoints-such as atezolizumab, avelumab, durvalumab, pembrolizumab, nivolumab, cemiplimab, tremelimumab, ipilimumab-in the fight against difficult to treat TNBCs as monotherapy as well as in more advanced combination strategies.
    Keywords:  immunotherapy; triple-negative breast cancer
    DOI:  https://doi.org/10.3390/cancers14163860
  8. Front Immunol. 2022 ;13 951143
    CAR T cells targeting the ganglioside NGcGM3 control ovarian tumors in the absence of toxicity against healthy tissues.
    Elisabetta Cribioli, Greta Maria Paola Giordano Attianese, George Coukos, Melita Irving.
      Chimeric antigen receptor (CAR) T cells have emerged as a powerful immunotherapeutic tool against certain hematological malignancies but a significant proportion of patients either do not respond or they relapse, sometimes as a result of target antigen loss. Moreover, limited clinical benefit has been reported for CAR therapy against epithelial derived solid tumors. A major reason for this is the paucity of solid tumor antigens identified to date that are broadly, homogeneously and stably expressed but not found on healthy tissues. To address this, here we describe the development and evaluation of CAR T cells directed against N-glycoslylated ganglioside monosialic 3 (NGcGM3). NGcGM3 derives from the enzymatic hydroxylation of N-acetylneuraminic acid (NAc) GM3 (NAcGM3) and it is present on the surface of a range of cancers including ovarian, breast, melanoma and lymphoma. However, while NAcGM3 is found on healthy human cells, NGcGM3 is not due to the 7deletion of an exon in the gene encoding for the enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Indeed, unlike for most mammals, in humans NGcGM3 is considered a neoantigen as its presence on tumors is the result of metabolic incorporation from dietary sources. Here, we have generated 3 CARs comprising different single chain variable fragments (scFvs) originating from the well-characterized monoclonal antibody (mAb) 14F7. We show reactivity of the CAR T cells against a range of patient tumor fragments and we demonstrate control of NGcGM3+ SKOV3 ovarian tumors in the absence of toxicity despite the expression of CMAH and presence of NGcGM3+ on healthy tissues in NSG mice. Taken together, our data indicate clinical potential for 14F7-based CAR T cells against a range of cancers, both in terms of efficacy and of patient safety.
    Keywords:  T cells; chimeric antigen receptor (CAR); ganglioside; immunotherapy; tumors
    DOI:  https://doi.org/10.3389/fimmu.2022.951143
  9. Nature. 2022 Aug 24.
    RASA2 ablation in T cells boosts antigen sensitivity and long-term function.
    Julia Carnevale, Eric Shifrut, Nupura Kale, William A Nyberg, Franziska Blaeschke, Yan Yi Chen, Zhongmei Li, Sagar P Bapat, Morgan E Diolaiti, Patrick O'Leary, Shane Vedova, Julia Belk, Bence Daniel, Theodore L Roth, Stefanie Bachl, Alejandro Allo Anido, Brooke Prinzing, Jorge Ibañez-Vega, Shannon Lange, Dalia Haydar, Marie Luetke-Eversloh, Maelys Born-Bony, Bindu Hegde, Scott Kogan, Tobias Feuchtinger, Hideho Okada, Ansuman T Satpathy, Kevin Shannon, Stephen Gottschalk, Justin Eyquem, Giedre Krenciute, Alan Ashworth, Alexander Marson.
      The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.
    DOI:  https://doi.org/10.1038/s41586-022-05126-w
  10. Nat Commun. 2022 Aug 23. 13(1): 4943
    scRNA-seq of gastric tumor shows complex intercellular interaction with an alternative T cell exhaustion trajectory.
    Keyong Sun, Runda Xu, Fuhai Ma, Naixue Yang, Yang Li, Xiaofeng Sun, Peng Jin, Wenzhe Kang, Lemei Jia, Jianping Xiong, Haitao Hu, Yantao Tian, Xun Lan.
      The tumor microenvironment (TME) in gastric cancer (GC) has been shown to be important for tumor control but the specific characteristics for GC are not fully appreciated. We generated an atlas of 166,533 cells from 10 GC patients with matched paratumor tissues and blood. Our results show tumor-associated stromal cells (TASCs) have upregulated activity of Wnt signaling and angiogenesis, and are negatively correlated with survival. Tumor-associated macrophages and LAMP3+ DCs are involved in mediating T cell activity and form intercellular interaction hubs with TASCs. Clonotype and trajectory analysis demonstrates that Tc17 (IL-17+CD8+ T cells) originate from tissue-resident memory T cells and can subsequently differentiate into exhausted T cells, suggesting an alternative pathway for T cell exhaustion. Our results indicate that IL17+ cells may promote tumor progression through IL17, IL22, and IL26 signaling, highlighting the possibility of targeting IL17+ cells and associated signaling pathways as a therapeutic strategy to treat GC.
    DOI:  https://doi.org/10.1038/s41467-022-32627-z
  11. Cytotherapy. 2022 Aug 23. pii: S1465-3249(22)00732-0. [Epub ahead of print]
    Emerging advances in engineered macrophages for tumor immunotherapy.
    Jing Hu, Qian Yang, Zhongyu Yue, Boting Liao, Huijuan Cheng, Wenqi Li, Honghua Zhang, Shuling Wang, Qingchang Tian.
      Macrophages are versatile antigen-presenting cells. Recent studies suggest that engineered modifications of macrophages may confer better tumor therapy. Genetic engineering of macrophages with specific chimeric antigen receptors offers new possibilities for treatment of solid tumors and has received significant attention. In vitro gene editing of macrophages and infusion into the body can inhibit the immunosuppressive effect of the tumor microenvironment in solid tumors. This strategy is flexible and can be applied to all stages of cancer treatment. In contrast, nongenetic engineering tools are used to block relevant signaling pathways in immunosuppressive responses. In addition, macrophages can be loaded with drugs and engineered into cellular drug delivery systems. Here, we analyze the effect of the chimeric antigen receptor platform on macrophages and other existing engineering modifications of macrophages, highlighting their status, challenges and future perspectives. Indeed, our analyses show that new approaches in the treatment of solid tumors will likely exploit macrophages, an innate immune cell.
    Keywords:  CAR-M; Drug-delivery system; Engineered macrophages; Gene editing; Immune check blocking therapy
    DOI:  https://doi.org/10.1016/j.jcyt.2022.07.001
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