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Use of chimeric antigen receptors (CARs), as an immune cell therapy, has generated excellent clinical outcomes against hematologic tumors in recent years. Among them, the CAR-NK (natural killer) therapy has shown better efficacy, and less toxicity, than chimeric antigen receptor T-cell (CAR-T) therapy. In our phase II clinical trials, administering chimeric costimulatory converting receptor (CCCR)-NK92 cells on advanced non-small cell lung cancer patients proved efficacious in cell and animal experiments. However, we observed occurrence of cytokine release syndrome (CRS), a rare and unexpected side effect, never reported before during CAR-NK therapy. Here, we provide a detailed report of the patient’s case, emphasize on the need to pay attention to CRS in NK cell therapy, and suggest improvements that will minimize potential toxicity.

Background Chimeric antigen receptor (CAR) engineered natural killer (NK) cells have shown their efficacy and superiority against cancer and possess the potential to become off-the-shelf immunotherapy products. Nonetheless, some challenges associated with CAR-NK cells still exist including inhibitory receptor engagement, antigen escape, and inadequate activation. Methods Given this, based on the concept of synthetic biology, we rationally designed a novel dual-targeted CAR (dtCAR), primarily comprising PD-L1 nanoantibody (PD-L1 Nb ) and NKG2D as the ectodomain, transmembrane and cytoplasmic domains (CP) of CD28, and the CP of 4-1BB and CD3ζ. NK92 cells were engineered to express this third-generation of dtCAR. We then elucidated the role of dtCAR-modified NK92 cells against cancer cells in vitro and in vivo. Results In vitro, the dtCAR-NK92 cells could still retain the characteristics of parental NK cells and exhibit improved NK cell cytotoxicity and produce more cytokines than NK92 cells when they were co-cultured with human lung cancer H1299 cells. Notably, the dtCAR-NK92 cell therapy might elicit clearance of H1299 cells by pyroptosis. Additionally, dtCAR-NK92 cells could considerably inhibit tumor growth in the human lung cancer H1299 cell tumor model. Conclusions We confirmed that expression of dtCAR enhanced NK92-cell activation and killing in vitro and in vivo, which provides a novel immunotherapeutic strategy for using NK-tailored CAR-engineered NK92 cells to treat human lung cancer.

•mVEGF165b as an immunization adjunct was able to induce high titers of anti-VEGF antibody.•High anti-VEGF antibody titers decrease immunosuppression by reducing the proportion of Tregs in the spleen or in peripheral circulation.•The decrease immunosuppression augments the immune response induced by peptide vaccine from Mucin-1.•mVEGF165b can be used as a potential adjunct with other tumor vaccines to increase anti-tumor efficacy and also promote the T cell infiltration into tumor. Compelling evidence has shown that blocking VEGF via monoclonal antibodies may be beneficial in that it not only inhibits tumor angiogenesis but also reduces immune suppression and promotes T cell infiltration into tumors. Herein, we determined whether our recently generated VEGF165b mutant could be used as a co-immunization adjunct to augment the peptide cancer-vaccine- induced immune response in a mouse model of breast cancer. When co-immunized mVEGF165b with the peptide-based cancer vaccine (MUC1, a T-cell epitope dominant peptide vaccine from Mucin1), the VEGF antibody titers increased approximately 600,000-fold in mice. Moreover, the anti-VEGF antibody also reduced the frequency of regulatory T cells (Tregs) in both preventive and therapeutic scenarios. Mechanistically, the decrease of the Tregs population was associated with a remarkably increased MUC-1-specific IFN-γ-producing CD8+ T cells and anti-MUC1 humoral response. Finally, this combination co-immunization produced a superior antitumor response and significantly prolonged survival of tumor-bearing mice. In conclusion, our findings suggest that mVEGF165b may be an ideal immunization adjunct to enhance the immune efficacy of peptide-based tumor vaccines by overcoming immune tolerance.

To address challenges such as the complex manufacturing of CAR-T and the immunosuppressive tumor microenvironment (TME), CAR-NK cells offer greater potential as an \"off-the-shelf\" therapy. To broaden tumor recognition and reduce the risk of immune escape associated with single-target approaches, we developed a single-promoter-driven multicistronic CAR-NK92 system that employs NKG2D for broad recognition of stress ligands and combines a PD1-CAR to reverse PD-L1 inhibitory signaling, thereby significantly enhancing antitumor efficacy. A multicistronic construct co-expressing PD1-CAR and NKG2D-CAR was generated using a P2A peptide under the control of a single CMV promoter and introduced into NK92 cells. The expression of PD-L1 and MICA/B was screened across multiple tumor cell lines, and the functional robustness of PN-CAR-NK92 cells was evaluated in all models through in vitro cytotoxicity and cytokine secretion assays. The in vivo translational efficacy was further validated using an H1299 xenograft model, with a direct comparison between PN-CAR-NK92 cells and NK92 cells. The multicistronic design enabled stable surface co-expression of both receptor modules, providing a structural basis for dual-target functionality. In vitro cytotoxicity assays demonstrated that PN-CAR-NK92 cells maintained robust antitumor activity across tumor cell lines with distinct PD-L1 and MICA/B expression profiles, whereas single-target CAR-NK92 cells displayed more restricted target specificity. These findings suggest that dual-target CAR engineering broadens antigen recognition coverage and may help reduce the limitations associated with single-target antigen dependence. Furthermore, PN-CAR-NK92 cells demonstrated significantly enhanced tumor suppression in the H1299 xenograft model compared with control groups. Dual-target PD1/NKG2D CAR-NK92 cells exhibit broadened antitumor activity across tumor cells with distinct ligand-expression profiles and may represent a promising strategy to reduce the limitations associated with single-target CAR therapies.

Vascular endothelial growth factor (VEGF) was considered as a critical growth factor for tumor expansion. The roles of VEGF121, VEGF165, and VEGF189 in tumor growth have been intensely investigated; however, involvements of another extracellular matrix (ECM)-binding VEGF isoform, namely VEGF183 (six amino acids shorter than VEGF189 in exon 6a), in physiological or pathological processes are still unclear although the wide tissue distribution. To investigate the role of VEGF183 in carcinogenesis, we generated murine breast cancer cell (EMT-6) clones stably overexpressing VEGF183, VEGF121, VEGF165, and VEGF189 shortened as V183, V121, V165, and V189, respectively. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) results showed that VEGF183, like all other VEGF-overexpressing isoforms except for VEGF121, could enhance the proliferation of mouse breast cancer EMT-6 cells. Immunochemistry results displayed that overexpressing VEGF183 and VEGF189 in EMT-6 cells induced larger proportional dilated microvessels. On the other hand, results from cell wound healing experiments demonstrated that all of the VEGF-overexpressing isoforms could increase the chemotaxis of EMT-6 cells in vitro. In conclusion, our results supported the idea that overexpression of VEGF183 promotes murine breast cancer cell proliferation in vitro and induces dilated intratumoral microvessels, and it plays a dissimilar role in comparison with that of VEGF189.

Clustered regularly interspaced short palindromic repeats (CRISPR) confer adaptive immunity in prokaryotes against viral infection and plasmid transformation. Here, we identified abundant clustered regularly interspaced homologous stem-loop pairs (CRIHSP) within human adaptive immunity-related genes, such as antigen receptor germline genes. By examining genomic stability under activation-induced cytidine deaminase (AID) overexpression, we found that CRIHSP structures are preferred targets for AID. Through collecting and analyzing cancer cell genomic mutation databases, we discovered that these mutation sites tend to cluster toward stem-loop structures. Importantly, the frequency of stem-loop sequences within immunoglobulin heavy chain variable (IGHV) gene segments was over 3-fold higher compared to that within other regions. We concurrently observed stem-loop structures frequently flanking RSS motifs. Conservative estimates indicate a 74.07% probability of immunoglobulin heavy chain diversity (IGHD) gene segment enclosure within stem-loop. These RSS-proximal stem-loops may assemble into homologous stem-loop pair (HSP)-like intermediates and subsequent CRIHSP arrays. Our results implicate CRIHSP architectures as critical enhancer of antigen receptor gene recombination and diversification. Further molecular dissection of these processes could potentially elucidate mechanisms underlying cancer cell genomic instability, chromatin folding and dynamic regulation, gene expression control, etc.

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer T/NK cells for the efficient expression of CARs. This study reported for the first time the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. Our results demonstrated that the selected CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered cells. This research will help to optimize the production of CAR-NK cells and improve their functional activity, to provide CAR-NK cell products with better and more uniform quality. Competing Interest Statement The authors have declared no competing interest.

The major challenge for universal chimeric antigen receptor T cell (UCAR-T) therapy is the inability to persist for a long time in patients leading to inferior efficacy clinically. The objective of this study was to design a novel UCAR-T cell that could avoid the occurrence of allo-rejection and provide effective resistance to allogeneic Natural Killer (NK) cell rejection, together with the validation of its safety and efficacy and . We prepared T-cell receptor (TCR), Human leukocyte antigen (HLA)-I/II triple-edited (TUCAR-T) cells and evaluated the anti-tumor efficacy ex vivo and in vivo. We measured the resistance of exogenous HLA-E expressing TUCAR-T (ETUCAR-T) to NK rejection by using an enhanced NK. Furthermore, we established the safety and efficacy of this regimen by treating Nalm6 tumor-bearing mice with a repeated high-dose infusion of ETUCAR-T. Moreover, we analyzed the effects of individual gene deficiency CAR-T on treated mice and the changes in the transcriptional profiles of different gene-edited T cells RNA-Seq. Data showed that HLA-II editing didn't impair the anti-tumor efficacy of TUCAR-T ex vivo and in vivo and we found for the first time that HLA-II deficiency could facilitate the persistence of CAR-T. Contrastively, as the most commonly eliminated target in UCAR-T, TCR deficiency was found to be a key disadvantageous factor for the shorter-term anti-tumor efficacy . Our study demonstrated ETUCAR-T could effectively resist allogeneic NK rejection and . Our research provided a potential and effective strategy for promoting the persistence of UCAR-T cells in clinical application. And it reveals the potential key factors of the poor persistence of UCAR-T along with new insights for future development.

CAR-T therapy has shown successful in the treatment of certain types of hematological malignancy, while the efficacy of CAR-T cell in treating solid tumors has been limited due to the exhaustion of CAR-T caused by the tumor microenvironment in solid tumors. Therefore, improving the exhaustion of CAR-T cell is one of the inspiring strategies for CAR-T treatment of solid tumors. As an important regulator in T cell immunity, the transcription factor RUNX3 not only negatively regulates the terminal differentiation T-bet gene, reducing the ultimate differentiation of T cells, but also increases the residency of T cells in non-lymphoid tissues and tumors. By overexpressing RUNX3 in CAR-T cells, we found that increasing the expression of RUNX3 maintained the low differentiation of CAR-T cells, further improving the exhaustion of CAR-T cells during antigen stimulation. In vitro, we found that RUNX3 could reduce the release of cytokines while maintaining CAR-T cells function. In re-challenge experiments, CAR-T cells overexpressing RUNX3 ( Runx3 -OE CAR-T) were safer than conventional CAR-T cells, while RUNX3 could also maintain the anti-tumor efficacy of CAR-T cells in vivo. Collectively, we found that Runx3 -OE CAR-T cells can improve CAR-T phenotype and reduce cytokines release while maintaining CAR-T cells function, which may improve the safety of CAR-T therapy in clinical trials.