Off-the-shelf CAR-NK cells targeting immunogenic cell death marker ERp57 execute robust antitumor activity and have a synergistic effect with ICD inducer oxaliplatin.

BACKGROUND: Chimeric antigen receptor natural killer (CAR-NK) therapy holds great promise for treating hematologic tumors, but its efficacy in solid tumors is limited owing to the lack of suitable targets and poor infiltration of engineered NK cells. Here, we explore whether immunogenic cell death (ICD) marker ERp57 translocated from endoplasmic reticulum to cell surface after drug treatment could be used as a target for CAR-NK therapy. METHODS: To target ERp57, a VHH phage display library was used for screening ERp57-targeted nanobodies (Nbs). A candidate Nb with high binding affinity to both human and mouse ERp57 was used for constructing CAR-NK cells. Various in vitro and in vivo studies were performed to assess the antitumor efficacy of the constructed CAR-NK cells. RESULTS: We demonstrate that the translocation of ERp57 can not only be induced by low-dose oxaliplatin (OXP) treatment but also is spontaneously expressed on the surface of various types of tumor cell lines. Our results show that G6-CAR-NK92 cells can effectively kill various tumor cell lines in vitro on which ERp57 is induced or intrinsically expressed, and also exhibit potent antitumor effects in cancer cell-derived xenograft and patient-derived xenograft mouse models. Additionally, the antitumor activity of G6-CAR-NK92 cells is synergistically enhanced by the low-dose ICD-inducible drug OXP. CONCLUSION: Collectively, our findings suggest that ERp57 can be leveraged as a new tumor antigen for CAR-NK targeting, and the resultant CAR-NK cells have the potential to be applied as a broad-spectrum immune cell therapy for various cancers by combining with ICD inducer drugs.

Cadherin 17 Nanobody-Mediated Near-Infrared-II Fluorescence Imaging-Guided Surgery and Immunotoxin Delivery for Colorectal Cancer.

Surgery and targeted therapy are of equal importance for colorectal cancer (CRC) treatment. However, complete CRC tumor resection remains challenging, and new targeted agents are also needed for efficient CRC treatment. Cadherin 17 (CDH17) is a membrane protein that is highly expressed in CRC and, therefore, is an ideal target for imaging-guided surgery and therapeutics. This study utilizes CDH17 nanobody (E8-Nb) with the near-infrared (NIR) fluorescent dye IRDye800CW to construct a NIR-II fluorescent probe, E8-Nb-IR800CW, and a Pseudomonas exotoxin (PE)-based immunotoxin, E8-Nb-PE38, to evaluate their performance for CRC imaging, imaging-guided precise tumor excision, and antitumor effects. Our results show that E8-Nb-IR800CW efficiently recognizes CDH17 in CRC cells and tumor tissues, produces high-quality NIR-II images for CRC tumors, and enables precise tumor removal guided by NIR-II imaging. Additionally, fluorescent imaging confirms the targeting ability and specificity of the immunotoxin toward CDH17-positive tumors, providing the direct visible evidence for immunotoxin therapy. E8-Nb-PE38 immunotoxin markedly delays the growth of CRC through the induction of apoptosis and immunogenic cell death (ICD) in multiple CRC tumor models. Furthermore, E8-Nb-PE38 combined with 5-FU exerts synergistically antitumor effects and extends survival. This study highlights CDH17 as a promising target for CRC imaging, imaging-guided surgery, and drug delivery. Nanobodies targeting CDH17 hold great potential to construct NIR-II fluorescent probes for surgery navigation, and PE-based toxins fused with CDH17 nanobodies represent a novel therapeutic strategy for CRC treatment. Further investigation is warranted to validate these findings for potential clinical translation.

A new design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency.

BACKGROUND: RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied. RESULTS: To explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system. CONCLUSIONS: We reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.