English
Abstract
MICB-targeting CAR-NK (chimeric antigen receptor-modified natural killer cells) therapy may serve as off-the-shelf immunotherapy. We designed soluble Anti-MICB-scFv blocks tumor immune evasion targeting the MICB antigen, thereby enhancing CAR-NK cytotoxicity while reactivating endogenous immune attacks against malignancies. The Anti-MICB-CAR includes two Anti-MICB-scFv connected by an F2A linker, the CD8 hinge and transmembrane domain, the 4-1BB co-stimulatory domain, the CD3ζ activation domain, and IL-15. The expression efficiency of Anti-MICB-CAR in NK cells was investigated by flow cytometry; ELISA demonstrated that Anti-MICB-CAR-NK secreted free Anti-MICB-scFv and detected IL-15 secretion. Flow cytometry and CCK8 were utilized to study Anti-MICB-CAR-NK on tumor cell viability. The PANC-1 xenograft model was established in order to elucidate the anti-tumor effects of Anti-MICB-CAR-NK in vivo. In vitro investigations have demonstrated that the treatment of tumor cells with Anti-MICB-CAR-NK supernatant + NK cells or Anti-MICB-CAR-NK cells not only significantly increased the cytotoxic activity of tumor cells, but also secreted and produced higher levels of IL-15, IFN-γ, TNF-α, perforin, and granzyme B compared with NK cells. Anti-MICB-CAR-NK cells exhibit strong cytotoxic activity against tumor cells with high MICB expression. In vivo, Anti-MICB-CAR-NK cells exhibited a substantial inhibitory effect on tumor growth. The IHC results reveal that Anti-MICB-CAR-NK cells show a more pronounced ability to infiltrate the tumor. We demonstrated the successful expression of Anti-MICB-CAR in NK cells, which enhances the anti-tumor activity of NK cells both in vitro and in vivo. This stress ligand-targeting approach provides a promising strategy for solid tumors.
Keywords:
MICB; IL-15; immunotherapy; CAR-NK; PANC-1
1. Introduction
In recent years, cellular immunotherapy has made significant strides in the field of oncology treatment, with the successful application of chimeric antigen receptor T cell (CAR-T) therapy in oncology being particularly notable. By genetically engineering T cells to target specific tumor antigens, CAR-T therapy has demonstrated significant efficacy in relapsed/refractory B-cell and lymphoma [1,2,3]. However, its efficacy in the treatment of solid tumors has fallen far short of expectations, mainly due to the immunosuppressive properties of the tumor microenvironment (TME), tumor antigen heterogeneity, and insufficient CAR-T cell infiltration [4,5,6]. These limitations have prompted researchers to explore more promising alternative cell therapies, among which natural killer cell (NK cell)-based CAR-NK has gradually become a research hotspot [7,8,9].
Compared with CAR-T cells, CAR-NK cells have unique advantages: NK cells are capable of recognizing and eliminating tumor cells without the need for human leukocyte antigen (HLA), thereby reducing the risk of graft-versus-host disease (GVHD) [10,11]. Furthermore, NK cells possess the capacity to engage multiple killing mechanisms through natural receptors (e.g., NKG2D), which may facilitate the overcoming of antigenic escape in solid tumors [12,13,14,15]. However, the development of CAR-NK therapy still faces many challenges, including difficulties in NK cell expansion in vitro, short survival time in vivo, and insufficient persistence of anti-tumor activity [16,17,18]. Solving these problems urgently requires multi-dimensional optimization in terms of target selection, cell engineering modification, and function enhancement strategies [19,20].
In recent years, solid tumor target screening has emerged as a novel research direction [21,22]. Claudin 18.2 is regarded as a viable target for immunotherapy in the treatment of solid tumors, including gastric cancer and pancreatic cancer. The clinical research results confirm that it shows good safety and promising anti-tumor activity in patients with refractory CLDN18.2 positive gastrointestinal tumors [23,24]. Mesothelin is expressed at high levels in a variety of tumors, including malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer. Conversely, its expression is low on the surface of normal pleura, peritoneum, and pericardium. Consequently, mesothelin is regarded as a promising target for cell therapy in the management of solid tumors. Lv, Jiang et al. demonstrated that the infusion of MSLN-CAR-T cells exhibited robust anti-tumor activity, thereby substantiating their potential as a therapeutic modality for GC [25]. EGFRvIII, a glioblastoma-associated antigen, has been identified as a target for immunotherapy. Gene amplification and/or mutations have been observed in approximately 50% of adult primary glioblastoma cases, rendering it an attractive candidate gene for targeted therapy. A clinical trial conducted at the University of Pennsylvania utilized anti-EGFRvIII CAR T cells to treat a cohort of 10 patients diagnosed with glioblastoma multiforme (GBM) that expressed the EGFRvIII protein. The study found this treatment to be well-tolerated and free from evidence of extratumoral toxicity, CRS, or cross-reactivity to wild-type EGFR [26]. Human epidermal growth factor receptor 2 (HER2) is overexpressed in many cancers and chimeric antigen receptor-T (CAR-T) cells targeting HER2 have been shown in clinical trials to be feasible and safe for treating recurrent or refractory central nervous system (CNS) tumors, potentially bringing new therapies for solid tumors [27]. Ganglioside 2 (GD2) is a surface glycolipid antigen that is highly expressed in neuroblastoma, astrocytoma, retinoblastoma, sarcoma, and melanoma, among other cancers. However, its expression is limited in normal tissues and primarily expressed at low levels on neuronal cell bodies and mesenchymal stem cells, rendering it an ideal target for CAR-T [28]. Sirtuins, a group of NAD+- dependent enzymes, have recently been identified as a key factor in the progression and resistance of pancreatic ductal adenocarcinoma (PDAC) and have the potential to become therapeutic targets. Among these, SIRT1 and SIRT2 have attracted the most research attention. The targeting of SIRT1 has been shown to have the potential to overcome drug resistance in invasive malignant tumors and improve treatment outcomes [29]. SIRT2 has been shown to inhibit mismatch repair (MMR) and promote immune escape in colorectal cancer by deacetylating MLH1. Moreover, high expression of SIRT2 is associated with reduced CD8+ T cell infiltration. Consequently, the targeting of SIRT2 has the potential to enhance immune cell infiltration into tumors [30]. In addition, sirtuins have been identified as biomarkers for prostate cancer (PCa) and have the potential to become therapeutic targets [31]. Sirtuins have been shown to play a unique role in the inhibition or promotion of tumor development. The prospect of targeting sirtuins in the context of cellular immunotherapy merits further investigation as a potential avenue for future research.
MICB (MHC class I chain-associated protein B), a stress-induced tumor-associated antigen, consists of α1, α2 (extracellular ligand-binding domain), and α3 (transmembrane/intracellular structural domain). MICB is highly expressed in a variety of solid tumors with restricted expression in normal tissues. It has been demonstrated that MICB binds to the NKG2D receptor of the α1-2 structural domain [32,33]. Not only does this activate the direct killing function of NK cells, but it also recruits CD8+ T cells, CD4+ T cells, and NK cells to co-infiltrate tumor tissues by modulating chemokine secretion in the tumor microenvironment (TME). In animal models, the MICB vaccine significantly increased the enrichment of CD8+ T cells (17.9-fold) and NK cells (38.9-fold) within the tumor, creating a synergistic attack by immune cells. This synergistic effect is particularly groundbreaking for drug-resistant tumors (e.g., MHC-I antigen-presentation-deficient tumors), where MICB, a highly conserved stress molecule, is widely expressed in different tumor types, giving cellular therapeutic oncology drugs targeting MICB antigens the potential to be “generalized”, bypassing the limitations of individualized antigens. Vaccines targeting the MICB antigen have shown significant inhibition of metastatic and drug-resistant tumors in both mouse and rhesus monkey models, with no significant side effects observed [34]. Kerry A Whalen et al. show that the MICA/B monoclonal antibody inhibited the shedding of MICA/B to promote innate immune cell-mediated anti-tumor activity [35]. Approved by the FDA, the antibody-(CLN-619) is currently undergoing Phase I clinical research. Mathieu Bléry et al. show that optimal tumor control was achieved with the MICA/B-ADC format in several solid tumor models, indicating that MICA and MICB are promising targets for cytotoxic immunotherapy [36].
Reducing the expression of MICB on the surface of tumor cells by hydrolyzing the α3 domain of MICB is one of the important mechanisms by which tumor cells conduct immune escape [37,38]. This mechanism provides a direction for the development of novel targeted drugs. Meanwhile, cytokine engineering provides a new idea to enhance the function of CAR-NK [39,40]. However, it will be difficult to activate other immune cells such as T cells, NK cells, and gamma delta T cells in the patient’s body solely with CAR-NK targeting MICB. Therefore, it is imperative to utilize MICB tumor targets to stimulate the activation of additional immune cells within the body, thereby initiating an attack on the tumor cells. Co-expression of IL-15 with CAR structure significantly promotes the survival, proliferation, and anti-tumor activity of NK cells and IL-15 not only maintains the persistence of CAR-NK through autocrine, but also remodels the tumor microenvironment and enhances immune cell infiltration and synergistic killing [41,42,43]. Based on this, we integrated an Anti-MICB-CAR-NK design that targets MICB and secretes IL-15 synergistically (Figure 1A). Firstly, Anti-MICB-scFv targets the MICB α3 domain to prevent MICB shedding, preventing tumor cells from immune escape and prompting the killing of tumor cells by MICB-CAR-NK. Secondly, the designed CAR structure can release free Anti-MICB-scFv, which can bind to MICB on the surface of tumor cells: this will newly reactivate NK and other relevant immune cells in vivo; modulate the secretion of chemokines in the tumor microenvironment (TME); and recruit CD8+ T cells, CD4+ T cells, and NK cells to co-infiltrate tumor tissues. Finally, CAR-NK expression of IL-15 significantly enhances CAR-NK expansion and therapeutic durability. We combine this unique stress ligand-targeting approach with NK cells and take advantage of the extensive high expression of MICB in solid tumors to design a targeted anti-tumor CAR-NK drug that will provide more anti-tumor properties than conventional NK cells, including broad solid tumor responsiveness.
Source: MDPI
Original link: https://www.mdpi.com/1422-0067/27/1/500
The FAI climbed 5.9 percent year-on-year in the first 11 months of 2018, quickening from the 5.7-percent growth in Jan-Oct, the National Bureau of Statistics (NBS) said Friday in an online statement.
The key indicator of investment, dubbed a major growth driver, hit the bottom in August and has since started to rebound steadily.
In the face of emerging economic challenges home and abroad, China has stepped up efforts to stabilize investment, in particular rolling out measures to motivate private investors and channel funds into infrastructure.
Friday's data showed private investment, accounting for more than 60 percent of the total FAI, expanded by a brisk 8.7 percent.
NBS spokesperson Mao Shengyong said funds into weak economic links registered rapid increases as investment in environmental protection and agriculture jumped 42 percent and 12.5 percent respectively, much faster than the average.
In breakdown, investment in high-tech and equipment manufacturing remained vigorous with 16.1-percent and 11.6-percent increases respectively in the first 11 months. Infrastructure investment gained 3.7 percent, staying flat. Investment in property development rose 9.7 percent, also unchanged.