HER2-targeting CAR-T cells show highly efficient anti-tumor activity against glioblastoma both in vitro and in vivo

HER2-targeting CAR-T cells show highly efficient anti-tumor activity against glioblastoma both in vitro and in vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article HER2-targeting CAR-T cells show highly efficient anti-tumor activity against glioblastoma both in vitro and in vivo Nianzhu Zhang, Xueying Li, Wenzhe Li, Lifen Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3926646/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 May, 2024 Read the published version in Genes & Immunity → Version 1 posted 12 You are reading this latest preprint version Abstract Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. Current treatment options for GBM include surgical resection, radiation, and chemotherapy, which predominantly only slow cancer growth and reduce symptoms, resulting in a 5-year survival rate of no more than 10%. Chimeric antigen receptor (CAR)-T cell therapy is a new class of cellular immunotherapies that has made great progress in the treatment of malignant tumors. Human epidermal growth factor receptor 2 (HER2) is over-expressed in GBM, and may provide a potential therapeutic target for GBM treatment. In this study, we constructed third-generation CAR-T cells targeting the HER2 antigen in GBM. HER2-CAR-T cells showed effective antitumor abilities both in vitro and in vivo . Furthermore, HER2-specific CAR-T cells exhibited strong cytotoxicity and cytokine-secreting ability against GBM cells in vitro . Anti-HER2 CAR-T cells also exerted increasing cytotoxicity from low to high effector-to-target (E: T) ratios. Importantly, anti-HER2 CAR-T cells delivered by peritumoral injection successfully stunted tumor progression in vivo . Moreover, peritumoral intravenous administration of anti-HER2 CAR-T exhibited a therapeutic improvement against GBM cells compared with intravenous administration. In conclusion, our studies show that HER2 CAR-T cells represent an emerging immunotherapy for the treatment of GBM. Biological sciences/Immunology/Immunotherapy/Immunosuppression Biological sciences/Immunology/Tumour immunology/Immunosurveillance/Immunoediting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Glioblastoma multiforme (GBM) is the most common type of malignant (cancerous) brain tumor. Glioblastoma can happen at any age, but it tends to occur more often in older adults and more often in men. All glioblastomas are grade IV brain tumors that contain the most abnormal-looking and most aggressive cells. Glioblastoma symptoms include headaches that keep getting worse, nausea and vomiting, blurred or double vision, and seizures. The disease’s five-year survival rate is only around 10%, with a median 15- to 18-month survival after diagnosis. Currently, standard treatments in the clinic include maximal safe surgical resection, radiation, and chemotherapy and are mostly limited by low therapeutic efficiency correlated with poor prognosis ( 1 ). Treatment is difficult since the tumor cells resist conventional therapies ( 2 ). Further, many drugs cannot cross the blood-brain barrier to act on the tumor ( 3 ). In addition, Solid tumors are heterotypic aggregates of many cell types. Consequently, all glioblastomas recur and grow quickly, invading and destroying healthy tissue. Thus, developing a novel for GBM is urgently needed. Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment ( 4 ). The CAR-T cells can effectively kill tumor cells by specifically recognizing and binding antigens on tumor cell membranes ( 5 ). CARs are engineered synthetic receptors that function to redirect lymphocytes, most commonly T cells, to recognize and eliminate cells, expressing a specific target antigen, in a manner independent of the major histocompatibility complex (MHC) ( 4 , 6 ). CAR-T immunotherapy is commonly used in hematological malignancies, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphoma, and multiple myeloma (MM) ( 7 ). Most currently, available CAR-T therapies target CD19 antigen ( 8 ). An ideal target should encompass a high level of surface expression, tissue specificity, and stability to ensure the effectiveness and tolerability of the CAR-T cells ( 9 ). The most frequently used target is CD19 on B-cells, predominantly used for the treatment of lymphoma and acute lymphocytic leukemia (ALL), leading to US Food and Drug Administration (FDA) approval of CAR-T cell therapies for clinical application. Another FDA-approved CAR-T cell-based therapy, which is targeted to B cell tumor antigens, is B cell maturation antigen (BCMA)-specific CAR-T, approved for the treatment of multiple myeloma. There are also other targets involved CD20, CD22, CD23, ROR1, CD4, CD30, CD33, GRP78, SLAMF and CD138. Until now, CAR-T cell therapy has achieved success in the context of hematological malignancies, but an increasing number of trials are also being conducted in solid tumor patients. There exist fundamental barriers to CAR-T therapy in solid tumors compared with hematological malignancies. One of the barriers to the effectiveness of cell therapy against solid tumors is antigen heterogeneity, which impairs the detection of cancer cells by T cells and reduces the impact of CAR-T therapy ( 10 ). The treatment of solid tumors is also limited by the inadequate transport and infiltration capability of CAR-T cells, due to the physical tumor barriers (such as the tumor stroma) that restrict the penetration and mobility of CAR-T cells ( 10 ). In addition to physical barriers, T cells must also confront highly immunosuppressive tumor microenvironments (TMEs) with cellular, molecular, and metabolic profiles that ultimately lead to T cell exhaustion and dysfunction ( 11 ). According to the above analysis, solid tumors may have cell-intrinsic resistance mechanisms to CAR-T cell cytotoxicity ( 12 ). Glioblastoma multiforme (GBM) remains incurable despite aggressive implementation of multimodal treatments after surgical debulking. Almost all GBM patients relapse within a narrow margin around the initial resected lesion due to post-surgery residual glioma stem cells (GSCs) ( 13 ). Due to the specific localization of tumors in the brain and the inherent resistance to conventional therapy, GBMs present unique treatment challenges. There is an urgent need to develop new strategies and novel delivery modalities for GBMs. Existing studies have thoroughly acknowledged the availability of utilizing CAR-T cells in the treatment of glioblastoma. CAR-T cell treatments targeting GBM have been the subject of many clinical studies. Currently, many targets, such as epidermal growth factor receptor variant III (EGFRvIII), interleukin (IL)–13Rα2, B7 homolog 3 protein (B7-H3, also known as CD276), CD70, ganglioside GD2, matrix metalloproteinase-2 (MMP-2) and natural killer group 2, member D (NKG2D), have been used in preclinical and clinical studies of CAR-T therapy conducted on GBM ( 14 ). The EGFR family member HER2, also known as ErbB2, is overexpressed in approximately 80% of GBM tumors ( 15 ). High expression of HER2 is associated with the development and progression of GBM ( 16 ). The ability of HER2-specific CAR-T cells to eliminate both differentiated GBM cells and GBM-initiating cells (GICs) makes it an attractive target tumor antigen (TA) ( 17 ). The traditional route of CAR-T treatment of GBM is through intravenous delivery. Several medications, including CAR-T, are prevented from entering the brain by the unique structure of the blood–brain barrier (BBB). When targeting HER2, the main focus is on the potential side effects caused by the expression of HER2 in various normal tissues, especially important organs ( 18 ). Therefore, a locoregional treatment strategy with better efficacy and prognosis for HER2-CAR-T cell-specific tumoricidal immunity treatment needs to be explored. In this study, HER2-targeted CAR-T (HER2-CAR-T) cells were engineered and their anti-tumor effects on GBM were evaluated both in vitro and in vivo. HER2-CAR-T cells exhibited strong cytotoxicity and cytokine-secreting efficiency against GBM cells in vitro . Furthermore, HER2-CAR-T cells delivered by peritumoral injection showed a higher therapeutical improvement against GBM cells than tail intravenous administration. Consequently, HER2-CAR-T cells showed high anti-tumor efficacy against GBM, suggesting that peritumoral injection has enormous potential to be an excellent CAR-T cell treatment strategy in the future. Materials and methods Construction of the HER2-, MSLN-, and EpCAM-targeting CAR vectors HER2-specific, MSLN-specific, and EpCAM-specific single-chain antibody fragments (scFvs) with a CD8 leading sequence, a CD8 hinge, and transmembrane sequence, as well as the intracellular signaling domain of 4-1BB, CD28, and CD3ζ in tandem, were engineered into third generation HER2-, MSLN-, and EpCAM-CAR T cells. The sequences, except for scFv, have been previously reported ( 19 ). The full-length nucleotide sequence was synthesized by Sangon Biotech (Shanghai, China), and was subsequently inserted into a CAR lentiviral expression vector [pCDH-EF1-MCS-EF1-puro] at two specific restriction enzyme sites (EcoR1 and Not1). Establishment of HER2-targeted CAR-T cells The pCDH-CMV-MCS-EF1-puro lentivirus system was used to generate a virus against HER2-targeted CAR. HEK293T cells were co-transfected with a HER2-targeting CAR vector, or control vector, with PLP1, PLP2, and PLP-VSVG, at a ratio of 23.1:16.5:16.5:9.9, polyethyleneimine (Polysciences, Warrington, PA, USA). Six hours after transfection, cells were re-fed with DMEM containing 20% FBS. After transfection, cell supernatant was collected at 48 h and 72 h, centrifuged at 4000 rpm for 5 min to remove cell debris, then filtered through a 0.45 µm filter. To concentrate the virus, 1: 4 PEG8000 (Sigma, Merk, Shanghai, China) was added and mixed every 30 minutes four times. After that, the virus was placed overnight at 4°C, then centrifuged at 4000 g for 30 min. The supernatant was removed, and lentivirus particles were resuspended in PBS and stored at -80°C. Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with a Ficoll kit (GE, Shanghai, China) and subsequently activated with anti-human CD3 (100 ng/mL; T&L Biotechnology) and anti-human CD28 (100 ng/mL; T&L Biotechnology). Recombinant human IL-2 (30 ng/mL; Novoprotein) and 1% penicillin–streptomycin (Gibco, Life Technologies, Shanghai, China) were added to X-VIVO 15 medium (Lonza, USA) for T cell proliferation. After activation for 24 h, T cells were transduced with HER2-targeted CAR lentiviral particles, selected with puromycin, and collected for subsequent in vivo and in vitro experiments after 12–14 days. Non-transduced T cells were used as a control group. Cell lines and culture conditions HOS, U118MG, U251, U87MG, HepG2, MKN-45, and SK-OV-3 cells were obtained from the American Type Culture Collection (ATCC, USA). HOS and SK-OV-3 cells were maintained in RPMI 1640 medium (Gibco, USA), and 293T, U118MG, U251, U87MG, HepG2, and MKN-45 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA). All cells were cultured in a medium supplemented with 10% FBS (BI, China), penicillin (60 µg/mL), and streptomycin (100 µg/mL) (Sangon, China) and maintained in an incubator with 5% CO 2 at 37°C. Immunohistochemistry Tissues of human glioblastoma were obtained from the Second Hospital of Dalian Medical University. Surgical glioblastoma tissues were fixed in 4% paraformaldehyde and embedded into paraffin. 4 µm thickness sections were deparaffinized in xylene and rehydrated with 100, 90, 80, and 70% ethanol to PBS. HER2 antibody (Cell Signaling Technology, USA) was utilized for immunostaining at room temperature (RT) for 2 hrs. After the slides were incubated with the HRP-labeled secondary Abs at RT for 1 h, 3,3' -diaminobenzidine (DAB) was added for coloration. The intensity of staining was analyzed by integrated optical density using Image-Pro R Plus software (version 6.0; Media Cybernetics, USA). Flow cytometry and antibodies Cells were incubated with antiCD16/CD32 (2.4G2) mAb to block Fcγ receptors. Recombinant anti-HER2 FITC-conjugated antibody and recombinant anti-EpCAM FITC-conjugated antibody (Sino Biological Inc., Beijing, China) were used to detect the expression of HER2 and EpCAM protein. Recombinant anti-mesothelin FITC antibody (Abcam, Cambridge, UK) was used to detect the expression of mesothelin. Cells were stained with HER2, EpCAM, or mesothelin antibody for 1h on ice. The expression of CAR on CAR-T cells was detected using biotinylated human HER2/MSLN/EpCAM (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA). Anti-mouse CD3 (17A2, BioLegend, USA) was used to detect the presence of CD3 + T cells in mouse peripheral blood. A FACS-Calibur (Becton Dickinson, USA) was used to perform flow cytometry according to prior guidelines ( 20 ), and FlowJo software (Tree Star) was utilized to analyze the data. Cytotoxicity assays Tumor cells were regarded as target cells (T) and suspended at a density (2 × 10 5 cells/ mL). Then, 0.1 mL cell suspension was transferred into a 96-well E-plate (ACEA Biosciences, Menlo Park, CA, USA) and cultured for 20 h. After that, HER2-targeting CAR-T cells (HER2-CAR-T) and untransfected T cells (NC-T) were regarded as effector cells (E) and added into each well separately at different E: T ratios (E: T of 5:1, or 2.5:1). The co-cultures were further cultured for the indicated times. RTCA software (xCELLigence RTCASP, ACEA, Los Angeles, CA, USA) was used to measure the viability of target cells in real time. Measurement of cytokine secretion HER2-CAR-T cells and NC-T cells were co-cultured with tumor cells for 24 h in a 96-well plate without any cytokines added. Enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, Grand Island, NY, USA) for specific cytokines (IFN-γ, TNF-α, GM-CSF, IL-6, and IL-8) were used to detect the level of cytokine production in the supernatant and assess the cell-killing efficacy. Tumor models and treatment 6- to 8-week-old NODPrkdcem26IL2rgem26/Nju (NCG) mice were obtained from NBRI (Nanjing Biomedical Research Institute of Nanjing University and Nanjing Galaxy Biopharma, Nanjing, China). Mice were maintained at 24 ± 1°C with free water and food intake, and illuminated for 12 hrs (08:00 to 20:00) in the specific pathogen-free (SPF) laboratory animal facility of Dalian Medical University (Dalian, China). For CDX (cell-derived xenograft, CDX) mouse models, 1 × 10 6 U118MG tumor cells in 100 µL of PBS were injected subcutaneously ( s.c. ) into the axilla of NCG mice. Tumor size was measured every 4 days. Mice were divided into 3 groups with 6 mice per group until the tumor volume reached 50–100 mm 3 . Tumor volumes were calculated according to the following formula: tumor volume = (length) × (width) 2 × 0.5, in which the length represented the longer dimension and tumor weights were recorded. Mice were monitored according to the Institutional Animal Care and Use Committee (IACUC) animal facilities rules and regulations. Situations when the experiment needed to be paused immediately were listed, as previously reported ( 21 ). For in vivo tumor killing, HER2-CAR-Ts were administered by two methods, peritumoral injection ( p.v. ) and intravenous injection ( i.v. ). Each U118MG-CDX mouse model was injected with 5 × 10 6 HER2-CAR-T cells in 200 µL PBS. Non-injected mice (non-transduced-T cells, named NC-T) were regarded as control groups. Statistical analysis Data are presented as mean ± standard deviation (SD) from at least three experiments. Two-tailed Student’s t-tests were performed to compare the statistical differences between groups, using GraphPad Prism software version 9. A p < 0.05 was considered to indicate statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Results Expression of HER2 in glioblastoma samples and tumor cell lines To understand the relationship between HER2 expression and glioblastoma, immunohistochemical analysis of glioma surgical sections was performed with HER2 antibody. Representative immunohistochemical results are shown in Fig. 1 A. HER2 was highly expressed in the glioma cells. To further determine whether HER2 could be a therapeutic target for tumor therapy, HER2-positive cancer cells were selected by flow cytometry. HER2 had high expression in a series of glioblastoma cells, U118MG, U251, and U87MG cells (Fig. 1 B). In addition, expression of HER2 in other types of tumor cells was also detected for subsequent research. Hepatocellular carcinoma cells (HepG2), human gastric carcinoma cells (MKN-45), and cells (SK-OV-3) all had high HER2 protein expression (Fig. 1 B). Human osteosarcoma cells (HOS) cells were used as negative controls (Fig. 1 B). Collectively, these data suggested a widespread expression of HER2 on different tumor cell types. Construction of third-generation CART cells targeting HER2 Given the importance of HER2 in glioblastoma and also other tumors, we generated a lentiviral expression plasmid of CAR targeting human HER2 by genetic engineering. HER2-CARs include three main parts: an extracellular antigen recognition domain of the single-chain Fragment variant (scFv) derived from an anti-HER2 antibody, a CD8 transmembrane domain, and an intracellular T cell activation domain of CD3ζ. Another two vital targets, EpCAM and MSLN were also constructed in the same way for further research. Costimulatory domains included both CD28 and 4-1BB to construct CD3ζ-CD28-41BB (Fig. 2 A). Lentivirus was produced by co-transfecting 293T cells with the HER2 CAR plasmid, PLP1, PLP2 and PLP-VSVG. HER2, EpCAM, and MSLN CAR-T cells were prepared by lentiviral infection using PBMCs. The efficiency of infection was evaluated by flow cytometry using the anti-HER2, anti-EpCAM, and anti-MSLN antibodies (Fig. 2 B). Cytotoxicity of HER2 CART cells in glioblastoma To evaluate the cytotoxicity and specificity of CAR-T cells, based on high expression of HER2, but not other targets, different targeting CAR-T cells were co-cultured with U118MG cells. Anti-HER2 CAR-T, anti-EpCAM CAR-T, and anti-MSLN CAR-T cells were cultured for 10 days and then independently co-cultured with U118MG cells at an effector-to-target (E: T) ratio of 2.5:1 and 5:1, with the NC-T (non-transduced T cells) group used as the control group. Real-time cytotoxicity assays (RTCA) provide automated real-time data acquisition continuously to monitor CAR-T cell-mediated killing of cancer cells and gain deeper insights into the specificity, potency, persistence, and efficiency of CAR-T cells. As shown in Fig. 3 A, compared with anti-EpCAM CAR-T and anti-MSLN CAR-T, the efficiency of HER2-CAR-T presented a stronger cell-killing ability on U118MG cells. Moreover, the cell-killing effect at an E: T ratio of 5:1 was greater than that at a 2.5:1 E: T ratio (Fig. 3 A), indicating that HER2-CAR-T cells had a dose-dependent effect on tumor cell killing. The cell killing rate was also analyzed and shown in Fig. 3 B. It is shown that T cell-derived TNF-α and IFN-γ are required for T-cell mediated killing of established tumors ( 22 ). Next, we compared the cytokine release mediated by HER2-CAR-T and NC-T against HER2-positive glioblastoma cell lines. IFN-γ and TNF-α released in the supernatant were all present at high levels in HER2-CAR-T-targeted U118MG, U251, and U87-MG cells, compared with the corresponding NC-T targeted cells (Fig. 4 A- 4 C). These results demonstrate that the effect of HER2-CAR-T cells on tumors is target-specific. Anti-tumor efficacy of HER2 CAR-T cells in vivo Since HER2 CAR-T cells are cytotoxic toward HER2-positive tumor cells in vitro , we speculated that HER2-CAR-T cells could also play a prominent part in tumor cell killing in vivo. We first evaluated the influence of HER2-CAR-T cells on tumor cell killing in tumor-bearing mice via two means of injection (Fig. 5 A). NCG mice, inoculated subcutaneously with U118MG cells, were randomly divided into three groups (n = 8): an NC-T control group (NC-T), a peritumoral injection group (HER2-CAR-T- p.v. ), and an intravenous injection group (HER2-CAR-T- i.v. ). Once tumors reached 50 mm 3 , 5 × 10 6 effector cells were administered in different ways. Twenty days after effector cell injection, compared with the NC-T group, a significant tumor suppressive effect was observed both in the HER2-CAR-T- p.v. group and HER2-CAR-T- i.v. group (Fig. 5 B). Day 44 after CAR-T cell implantation, NC-T mice were euthanized due to the enormous volume of tumors (Fig. 5 B). The HER2-CAR-T- p.v. group and HER2-CAR-T- i.v. group were still fed to observe the influence of different injection modes of CAR-T cells on tumor therapy. On day 44, peripheral blood was collected from each mouse and the proportion of CD3 + T cells was measured. As illustrated in Fig. 5 C, the proportion of CD3 + T cells in peripheral blood was still at higher levels in the HER2-CAR-T- p.v. group and HER2 CAR-T- i.v. group, while in the NC-T group was depleted. To further investigate CAR-T cell efficacy, tumor tissue was obtained surgically, and CAR-T cells were measured via flow cytometry. Notably, the proportions of CAR-T cells were kept at high levels in the HER2-CAR-T- p.v. group and HER2-CAR-T- i.v. group compared with the NC-T group (Fig. 5 D). Interestingly, the HER2-CAR-T- p.v. group displayed a much higher proportion of tumor-infiltrating HER2-CAR-T cells in comparison with the HER2 CAR-T- i.v. group, implying a better efficiency of peritumoral administration towards glioma (Fig. 5 D). To further investigate the effect of CAR-T treatment on glioma, a long-term increase in tumor volume was measured under CAR-T therapy by p.v. and i.v. Tumor volume was smaller in the HER2-CAR-T- p.v. group, but was not statistically significant until day 72 (Fig. 5 B). We subsequently measured GM-CSF (granulocyte-macrophage colony-stimulating factor) and IFN-γ in the serum of HER2-CAR-T- p.v. and HER2-CAR-T- i.v. mice (Fig. 5 E). Elevated GM-CSF and IFN-γ were found in the HER2-CAR-T mice, especially in HER2-CAR-T- p.v. mice, while IL-8 and IL-6 exhibited decreased expression (Fig. 5 F). Taken together, our results suggest that CAR-T therapy by p.v. exerts a stronger therapeutic effect on gliomata compared with i.v. administration. Based on these results, we propose that HER2-CAR-T therapy could be considered as a rational immunotherapeutic strategy, and peritumoral administration could be an innovative and locoregional approach for GBM. Discussion In our study, we found an anti-tumor activity of HER2-CAR-T cells against HER2-positive GBM cells, as well as other HER2-positive tumor cell types. However, high tumor heterogeneity, local physical barriers, hostile tumor microenvironment, and antigen escape still make CAR-T therapy a challenging treatment ( 23 ). The survival of GBM cells in the harsh local environment of the brain and the elimination of multiple types of tumor antigens also pose unique difficulties that need to be solved. Tumor-associated antigens (TAA) are the main targets for CAR-T-engineered therapy ( 24 ). The critical therapeutic barrier is the diverse expression of TAAs of GBM cells. Moreover, apart from HER2, various expression levels of other antigens in GBM also impair the function of HER2-CAR-T because the diversity of GBM cell antigens makes it impossible to identify HER2 antigen, thereby affecting the efficiency of tumor treatment. Till today, there has been a series of CAR-T therapeutic targets, towards GBM, involving EGFRvIII, IL13Ra2, HER2, B7-H3, CD70, GD2, MMP2, and NKG2D ( 14 ). To explore the targeting of multiple TAAs on GBM by identified CAR-T cells, including the co-expression of several CARs on a single T cell, and expression of a chimeric receptor including two or more antigen recognition domains, which in turn leads to the identification of multiple antigens through individual receptors ( 25 ) are both more effective treatment for GMB to conquer the tumor heterogeneity. Combining CAR-T treatment with the other immunotherapies was also a meaningful strategy for tumor therapy. In hematological malignancy, a combination of PD-1 blockade and CD19 CAR-T cell therapy in B-ALL patients improved outcomes and improved CAR-T cell persistence ( 26 ). A recent study has shown that third-generation HER2-specific CAR-T cells can efficiently eliminate GBM cells in vitro and that the activity of the administered CAR-T cells is increased by their combination with PD-1 blockade ( 27 ). Before CAR-T cells can get to work, they must bypass the blood-brain barrier (BBB), a non-fenestrated physical barrier comprised of specialized capillary endothelial cells interconnected by multi-protein tight junctions ( 28 ). Unlike hematological malignancies, the peripheral blood is not the compartment of therapeutic action, and the effective CAR-T cell dose and frequency/schedule of administration are elusive in GBM. In addition to the above, the major concern when targeting HER2 is potential side effects due to HER2 expression in various normal tissues, especially in vital organs, although this has rarely been an issue with HER2-specific CAR-T cell administration in humans thus far. During CAR-T treatment, a high accumulation of CAR-T cells occurs in normal lung and abdominal/mediastinal lymph nodes, on which HER2 is expressed, although at a low level ( 27 ), which could reduce the efficiency of tumor treatment. Higher infusion of a large number of CAR-T cells can trigger the release of life-threatening supraphysiological levels of pro-inflammatory cytokines which could cause serious side effects and disorders. Taken together, the innovation of CAR-T cell delivery is extremely critical for GBM therapy. We establish a peritumoral injection strategy of CAR-T in a CDX mouse model. Compared with the traditional approach using intravenous injection, we show peritumoral injection of CAR-T cells more efficiently primes locoregional immunity for GBM therapy. Furthermore, CAR-T therapy via peritumoral injection can be combined with novel strategies to enhance CAR-T cell cytotoxic ability to GBM. Currently, CAR-T cells can be classified into four different generations, with next or fifth-generation CARs currently under active development ( 25 ). Here, engineered third-generation CAR-T cells were utilized, which combined 4-1BB and CD28 signaling domains to provide superior activation and proliferation capacity compared with second-generation CAR-T cells. However, overstimulation of T-cell activity by two costimulatory molecules can induce sharp increases in cytokine secretion, leading to cytokine release syndrome (CRS). Recently, the original CD3ζ has been replaced with three peptide chains of CD3, γ, δ, and ε which can be used to solve the problem of T cell depletion, exhaustion, and CRS during CAR-T treatment ( 29 ). Among all CAR activations, BB-ζ secreted the highest level of cytokines, and a large portion of cytokines was related to CRS, suggesting that the use of CAR with other three peptide chains could reduce the occurrence of CRS and enhance the safety of CAR-T therapy ( 29 ). A CAR that uses one of the signaling domains of another peptide chain rather than the ζ chain could mitigate or prevent the shortcomings of existing CAR-T cell therapies ( 29 ). Further research will focus on the therapeutic efficiency of the other peptide chains of CD3. Fourth-generation CAR-T cells incorporate cytokines or co-stimulatory ligands to further enhance the T cell response, or suicide genes to cause the CAR-T cells to self-destruct if needed. Regulatory elements of suicide genes could increase the safety and targeting of CAR-T therapy. Fourth-generation CAR-T cells, redirected for universal cytokine killing, could secrete specific cytokines (currently mainly IL-12) in the tumor region, thereby modifying the tumor microenvironment, and recruiting and activating other immune cells for an immune response. Fifth-generation CAR-T technology will break through individual limitations to be universal and produced on a large-scale, and enable treatment among different individuals. The development of new CAR-T therapies should therefore consider strategies that can achieve a more balanced immune response. Declarations Conflict of interest The authors declare no commercial or financial conflict of interest. Author contributions Zhang NZ designed the manuscript; Li XY wrote the manuscript; Zhao LF prepared the figures; Li WZ revised the manuscript. All authors have read and approved the final manuscript. Acknowledgements This work was Supported by the National Natural Science Foundation of China, No. 32171279; Natural Science Foundation of Liaoning Province, No. 2022-BS-254. References Huang B, Li X, Li Y, Zhang J, Zong Z, Zhang H. Current Immunotherapies for Glioblastoma Multiforme. Frontiers in immunology. 2020;11:603911. Yalamarty SSK, Filipczak N, Li X, Subhan MA, Parveen F, Ataide JA, et al. Mechanisms of Resistance and Current Treatment Options for Glioblastoma Multiforme (GBM). Cancers. 2023;15(7). Pardridge WM. Drug transport across the blood-brain barrier. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 2012;32(11):1959–72. Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood cancer journal. 2021;11(4):69. Gorchakov AA, Kulemzin SV, Kochneva GV, Taranin AV. Challenges and Prospects of Chimeric Antigen Receptor T-cell Therapy for Metastatic Prostate Cancer. European urology. 2020;77(3):299–308. Zhao Z, Chen Y, Francisco NM, Zhang Y, Wu M. The application of CAR-T cell therapy in hematological malignancies: advantages and challenges. Acta pharmaceutica Sinica B. 2018;8(4):539–51. Landoni E, Savoldo B. Treating hematological malignancies with cell therapy: where are we now? Expert opinion on biological therapy. 2018;18(1):65–75. Savani M, Oluwole O, Dholaria B. New targets for CAR T therapy in hematologic malignancies. Best practice & research Clinical haematology. 2021;34(3):101277. Wei J, Han X, Bo J, Han W. Target selection for CAR-T therapy. Journal of hematology & oncology. 2019;12(1):62. Marofi F, Motavalli R, Safonov VA, Thangavelu L, Yumashev AV, Alexander M, et al. CAR T cells in solid tumors: challenges and opportunities. Stem cell research & therapy. 2021;12(1):81. Hou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nature reviews Drug discovery. 2021;20(7):531–50. Larson RC, Kann MC, Bailey SR, Haradhvala NJ, Llopis PM, Bouffard AA, et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature. 2022;604(7906):563–70. Chen C, Jing W, Chen Y, Wang G, Abdalla M, Gao L, et al. Intracavity generation of glioma stem cell-specific CAR macrophages primes locoregional immunity for postoperative glioblastoma therapy. Science translational medicine. 2022;14(656):eabn1128. Wang C, Li Y, Gu L, Chen R, Zhu H, Zhang X, et al. Gene Targets of CAR-T Cell Therapy for Glioblastoma. Cancers. 2023;15(8). Mineo JF, Bordron A, Baroncini M, Maurage CA, Ramirez C, Siminski RM, et al. Low HER2-expressing glioblastomas are more often secondary to anaplastic transformation of low-grade glioma. Journal of neuro-oncology. 2007;85(3):281–7. Hynes NE, MacDonald G. ErbB receptors and signaling pathways in cancer. Current opinion in cell biology. 2009;21(2):177–84. Ahmed N, Salsman VS, Kew Y, Shaffer D, Powell S, Zhang YJ, et al. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors. Clinical cancer research: an official journal of the American Association for Cancer Research. 2010;16(2):474–85. Press MF, Cordon-Cardo C, Slamon DJ. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene. 1990;5(7):953–62. Zhang Q, Liu G, Liu J, Yang M, Fu J, Liu G, et al. The antitumor capacity of mesothelin-CAR-T cells in targeting solid tumors in mice. Molecular therapy oncolytics. 2021;20:556–68. Cossarizza A, Chang HD, Radbruch A, Acs A, Adam D, Adam-Klages S, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). European journal of immunology. 2019;49(10):1457–973. Zhang Q, Zhang Z, Liu G, Li D, Gu Z, Zhang L, et al. B7-H3 targeted CAR-T cells show highly efficient anti-tumor function against osteosarcoma both in vitro and in vivo. BMC cancer. 2022;22(1):1124. Zhang B, Karrison T, Rowley DA, Schreiber H. IFN-gamma- and TNF-dependent bystander eradication of antigen-loss variants in established mouse cancers. The Journal of clinical investigation. 2008;118(4):1398–404. Kankeu Fonkoua LA, Sirpilla O, Sakemura R, Siegler EL, Kenderian SS. CAR T cell therapy and the tumor microenvironment: Current challenges and opportunities. Molecular therapy oncolytics. 2022;25:69–77. Liu D. Cancer biomarkers for targeted therapy. Biomarker research. 2019;7:25. Khan JF, Khan AS, Brentjens RJ. Application of CAR T cells for the treatment of solid tumors. Progress in molecular biology and translational science. 2019;164:293–327. Song W, Zhang M. Use of CAR-T cell therapy, PD-1 blockade, and their combination for the treatment of hematological malignancies. Clinical immunology (Orlando, Fla). 2020;214:108382. Maggs L, Cattaneo G, Dal AE, Moghaddam AS, Ferrone S. CAR T Cell-Based Immunotherapy for the Treatment of Glioblastoma. Frontiers in neuroscience. 2021;15:662064. Ballabh P, Braun A, Nedergaard M. The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiology of disease. 2004;16(1):1–13. Velasco Cárdenas RM, Brandl SM, Meléndez AV, Schlaak AE, Buschky A, Peters T, et al. Harnessing CD3 diversity to optimize CAR T cells. Nature immunology. 2023;24(12):2135–49. Additional Declarations There is NO conflict of interest to disclose. The authors declare no commercial or financial conflict of interest. Cite Share Download PDF Status: Published Journal Publication published 03 May, 2024 Read the published version in Genes & Immunity → Version 1 posted Editorial decision: revise 04 Mar, 2024 Review # 3 received at journal 25 Feb, 2024 Review # 1 received at journal 24 Feb, 2024 Review # 2 received at journal 21 Feb, 2024 Reviewer # 3 agreed at journal 16 Feb, 2024 Reviewer # 2 agreed at journal 10 Feb, 2024 Reviewer # 1 agreed at journal 10 Feb, 2024 Reviewers invited by journal 10 Feb, 2024 Submission checks completed at journal 06 Feb, 2024 First submitted to journal 05 Feb, 2024 Unknown event 05 Feb, 2024 Editor assigned by journal 04 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3926646","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":271395070,"identity":"fc822de9-d49d-4013-9c57-dba9d9e4105c","order_by":0,"name":"Nianzhu Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYDACCTjJw8DwwcCGh5+Z+fADorUwzqhIk5NsZ0szIEILA1gLM8+Zw8YG53kUJPBoYJCf3fzs4dcdFvLm/GePSfC2MSduPszDYMBQYxONSwvjnGPmxrJnJAx3NpxLk5BsY0vcdpj3wAOGY2m5DTi0MEskmElLtkkwbjjYYyZh2MYD1MKXYMDYcBinFjaJ9G8gLfYbDvOYSSS2SSRubuYxkMCnhUcix0zyI1DlhmNALQfOGBgbMBPQIiGRUybN2CaRvOEMj7FlQ0WCnMRhYCAn4PGL/Iz0bZI/2+psN5w/Y3j7j8F/Hv7+w4cffKixwakFHAQ8GEIJeJSDAOMPAgpGwSgYBaNghAMASG9WVPoGNHMAAAAASUVORK5CYII=","orcid":"","institution":"The Second Hospital of Dalian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Nianzhu","middleName":"","lastName":"Zhang","suffix":""},{"id":271395071,"identity":"25bfce46-0cb6-4078-90bf-b574a3e1a3cb","order_by":1,"name":"Xueying Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xueying","middleName":"","lastName":"Li","suffix":""},{"id":271395072,"identity":"19bcf8f4-cb62-4405-80b9-d41b032b95c8","order_by":2,"name":"Wenzhe Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenzhe","middleName":"","lastName":"Li","suffix":""},{"id":271395073,"identity":"52c9703b-0df8-4de4-b796-60071de279b0","order_by":3,"name":"Lifen Zhao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lifen","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-02-04 07:20:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3926646/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3926646/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41435-024-00275-6","type":"published","date":"2024-05-03T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51083759,"identity":"5b8b44f5-3fa6-4dd0-91d1-9abb10629efd","added_by":"auto","created_at":"2024-02-13 19:42:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":608758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of HER2 in GBM samples and tumor cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative images of immunohistochemical analysis showing HER2 levels in tumor and adjacent tissue from GBM patients. After washing, secondary antibody was incubated and visualized with DAB. Light yellow indicates low expression and deep yellow indicates high expression. GBM, glioblastoma. scale bar=50 μm and 100 μm.\u003c/p\u003e\n\u003cp\u003eB. Surface expression of HER2 on tumor cell lines (U118MG, U251, U87MG, HepG2, MKN-45, SK-OV-3, and HOS). HER2 expression on the cell surface was verified by flow cytometry. Numbers indicate the percentage of HER2\u003csup\u003e+\u003c/sup\u003e cells, and 10,000 events were acquired for each analysis.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3926646/v1/99c9ce5825a65cd5e0fcf18f.png"},{"id":51083761,"identity":"71581749-bab4-4aa3-980b-1a2783a68f5e","added_by":"auto","created_at":"2024-02-13 19:42:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of 3rd-generation CAR-T cells targeting HER2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic diagram of the HER2 CAR-T transgene.\u003c/p\u003e\n\u003cp\u003eB. HER2, EpCAM, and MSLN expression were measured by flow cytometry. CAR-T cells were incubated with biotinylated anti-HER2, anti-MSLN, and anti-EpCAM, followed by incubation with APC streptavidin (1:1000). ***p \u0026lt; 0.001 [Student’s t-test (unpaired)]. Data (B) is shown as mean ± SD of triplicates, and is representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3926646/v1/fcb7628e4b36a8794cbd3862.png"},{"id":51083762,"identity":"fe53a50e-20ef-4fcd-86b0-46e3bec4a9d4","added_by":"auto","created_at":"2024-02-13 19:42:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":166308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional study of HER2 CAR-T cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Cytotoxicity of CAR-T cells against solid tumor cell lines was analyzed by RTCA assay. HER2-CAR-T cells, EpCAM-CAR-T cells, and MSLN-CAR-T cells were all used as effector cells. Cytotoxicity of HER2-, EpCAM-, and MSLN-CAR-T cells, and NCT cells against U118MG cells at an E: T of 5:1 and 2.5:1 for 80 h were analyzed. Black arrow represents the time when effector cells were added.\u003c/p\u003e\n\u003cp\u003eB. Comparison of killing rates under different effector cells and E: T ratios on U118MG cells. NS, not significant, *p \u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 [Student’s t-test (unpaired)]. Data are shown as mean ± SD of triplicates and are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3926646/v1/4ac0d42790568ac806897c64.png"},{"id":51083763,"identity":"b2e4bafb-f0b7-4532-817d-0ed5c75c669c","added_by":"auto","created_at":"2024-02-13 19:42:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":96042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHER2 CAR-T cell-mediated cytokine release.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Levels of IFN-γ and TNF-α released by HER2 CAR-T cells were analyzed by ELISA after incubation with U118MG cells at an E: T of 5:1 and 2.5:1 for 20 h.\u003c/p\u003e\n\u003cp\u003eB and C. Levels of IFN-γ and TNF-α released by HER2 CAR-T cells analyzed by ELISA after incubation with another two glioma cell lines, U251 (B) and U87MG (C) cells at an E: T of 2.5:1 for 20 h. NS, not significant, *p \u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 [Student’s t-test (unpaired)]. Data are shown as mean ± SD of triplicates, and are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3926646/v1/c596031e86d2c6c9c85be50b.png"},{"id":51083764,"identity":"7b9cad76-856f-4360-9e0e-7469da74ed27","added_by":"auto","created_at":"2024-02-13 19:42:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":214365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-tumor efficacy of HER2-CAR-T cells against transplanted U118MG cell tumors in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Experimental outline. NCG mice were divided into 3 groups with 6 mice per group. Mice were injected \u003cem\u003es.c.\u003c/em\u003e with 1 × 10\u003csup\u003e6\u003c/sup\u003e U118MG cells. Once tumors were well established on day 20, xenograft-bearing mice received 5 × 10\u003csup\u003e6\u003c/sup\u003e NC-T cells and CAR-T cells. CAR-T cells were injected either \u003cem\u003ep.v.\u003c/em\u003e or \u003cem\u003ei.v.\u003c/em\u003e Mice in the NC-T group were sacrificed on Day 44 while mice in CAR-T group were sacrificed on Day 72. Tumor size was measured every 4 days.\u003c/p\u003e\n\u003cp\u003eB. Presence of CD3\u003csup\u003e+\u003c/sup\u003e T cells in mouse peripheral blood 44 days after CAR-T cell injection. Peripheral blood mononuclear cells (PBMC) were isolated and stained with anti-CD3 antibody.\u003c/p\u003e\n\u003cp\u003eC. Absence of CAR-T cells in mouse tumors 44 days after injection. Tumor tissue was digested and CD3 and HER2 double-positive cells were analyzed by polychromatic flow cytometry. Cells were probed with anti-CD3 and anti-HER2.\u003c/p\u003e\n\u003cp\u003eD. GM-CSF and IFN-γ released by HER2 CAR-T cells and NC-T cells were determined by ELISA 44 days after injection.\u003c/p\u003e\n\u003cp\u003eE. IL-8 and IL-6 released by HER2 CAR-T cells and NC-T cells analyzed by ELISA 44 days after injection. NS, not significant, *p \u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001 [Student’s t-test (unpaired)]. Data are shown as mean ± SD of triplicates, and are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3926646/v1/3fc3e057ea6b6da45dc9f1b1.png"},{"id":55823221,"identity":"1b37e0e9-ed10-499f-a208-a237d384cf0e","added_by":"auto","created_at":"2024-05-04 00:46:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1411427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3926646/v1/790f3977-f0a9-49e9-8514-0f5be50767ce.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.\nThe authors declare no commercial or financial conflict of interest.","formattedTitle":"HER2-targeting CAR-T cells show highly efficient anti-tumor activity against glioblastoma both in vitro and in vivo","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioblastoma multiforme (GBM) is the most common type of malignant (cancerous) brain tumor. Glioblastoma can happen at any age, but it tends to occur more often in older adults and more often in men. All glioblastomas are grade IV brain tumors that contain the most abnormal-looking and most aggressive cells. Glioblastoma symptoms include headaches that keep getting worse, nausea and vomiting, blurred or double vision, and seizures. The disease\u0026rsquo;s five-year survival rate is only around 10%, with a median 15- to 18-month survival after diagnosis. Currently, standard treatments in the clinic include maximal safe surgical resection, radiation, and chemotherapy and are mostly limited by low therapeutic efficiency correlated with poor prognosis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Treatment is difficult since the tumor cells resist conventional therapies (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Further, many drugs cannot cross the blood-brain barrier to act on the tumor (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In addition, Solid tumors are heterotypic aggregates of many cell types. Consequently, all glioblastomas recur and grow quickly, invading and destroying healthy tissue. Thus, developing a novel for GBM is urgently needed.\u003c/p\u003e \u003cp\u003eChimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The CAR-T cells can effectively kill tumor cells by specifically recognizing and binding antigens on tumor cell membranes (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). CARs are engineered synthetic receptors that function to redirect lymphocytes, most commonly T cells, to recognize and eliminate cells, expressing a specific target antigen, in a manner independent of the major histocompatibility complex (MHC) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). CAR-T immunotherapy is commonly used in hematological malignancies, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), lymphoma, and multiple myeloma (MM) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Most currently, available CAR-T therapies target CD19 antigen (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). An ideal target should encompass a high level of surface expression, tissue specificity, and stability to ensure the effectiveness and tolerability of the CAR-T cells (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The most frequently used target is CD19 on B-cells, predominantly used for the treatment of lymphoma and acute lymphocytic leukemia (ALL), leading to US Food and Drug Administration (FDA) approval of CAR-T cell therapies for clinical application. Another FDA-approved CAR-T cell-based therapy, which is targeted to B cell tumor antigens, is B cell maturation antigen (BCMA)-specific CAR-T, approved for the treatment of multiple myeloma. There are also other targets involved CD20, CD22, CD23, ROR1, CD4, CD30, CD33, GRP78, SLAMF and CD138. Until now, CAR-T cell therapy has achieved success in the context of hematological malignancies, but an increasing number of trials are also being conducted in solid tumor patients. There exist fundamental barriers to CAR-T therapy in solid tumors compared with hematological malignancies. One of the barriers to the effectiveness of cell therapy against solid tumors is antigen heterogeneity, which impairs the detection of cancer cells by T cells and reduces the impact of CAR-T therapy (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The treatment of solid tumors is also limited by the inadequate transport and infiltration capability of CAR-T cells, due to the physical tumor barriers (such as the tumor stroma) that restrict the penetration and mobility of CAR-T cells (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In addition to physical barriers, T cells must also confront highly immunosuppressive tumor microenvironments (TMEs) with cellular, molecular, and metabolic profiles that ultimately lead to T cell exhaustion and dysfunction (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). According to the above analysis, solid tumors may have cell-intrinsic resistance mechanisms to CAR-T cell cytotoxicity (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlioblastoma multiforme (GBM) remains incurable despite aggressive implementation of multimodal treatments after surgical debulking. Almost all GBM patients relapse within a narrow margin around the initial resected lesion due to post-surgery residual glioma stem cells (GSCs) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Due to the specific localization of tumors in the brain and the inherent resistance to conventional therapy, GBMs present unique treatment challenges. There is an urgent need to develop new strategies and novel delivery modalities for GBMs. Existing studies have thoroughly acknowledged the availability of utilizing CAR-T cells in the treatment of glioblastoma. CAR-T cell treatments targeting GBM have been the subject of many clinical studies. Currently, many targets, such as epidermal growth factor receptor variant III (EGFRvIII), interleukin (IL)\u0026ndash;13Rα2, B7 homolog 3 protein (B7-H3, also known as CD276), CD70, ganglioside GD2, matrix\u0026ensp;metalloproteinase-2\u0026ensp;(MMP-2) and natural killer group 2, member D (NKG2D), have been used in preclinical and clinical studies of CAR-T therapy conducted on GBM (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The EGFR family member HER2, also known as ErbB2, is overexpressed in approximately 80% of GBM tumors (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). High expression of HER2 is associated with the development and progression of GBM (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The ability of HER2-specific CAR-T cells to eliminate both differentiated GBM cells and GBM-initiating cells (GICs) makes it an attractive target tumor antigen (TA) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The traditional route of CAR-T treatment of GBM is through intravenous delivery. Several medications, including CAR-T, are prevented from entering the brain by the unique structure of the blood\u0026ndash;brain barrier (BBB). When targeting HER2, the main focus is on the potential side effects caused by the expression of HER2 in various normal tissues, especially important organs (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Therefore, a locoregional treatment strategy with better efficacy and prognosis for HER2-CAR-T cell-specific tumoricidal immunity treatment needs to be explored.\u003c/p\u003e \u003cp\u003eIn this study, HER2-targeted CAR-T (HER2-CAR-T) cells were engineered and their anti-tumor effects on GBM were evaluated both \u003cem\u003ein vitro\u003c/em\u003e and in vivo. HER2-CAR-T cells exhibited strong cytotoxicity and cytokine-secreting efficiency against GBM cells \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, HER2-CAR-T cells delivered by peritumoral injection showed a higher therapeutical improvement against GBM cells than tail intravenous administration. Consequently, HER2-CAR-T cells showed high anti-tumor efficacy against GBM, suggesting that peritumoral injection has enormous potential to be an excellent CAR-T cell treatment strategy in the future.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of the HER2-, MSLN-, and EpCAM-targeting CAR vectors\u003c/h2\u003e \u003cp\u003eHER2-specific, MSLN-specific, and EpCAM-specific single-chain antibody fragments (scFvs) with a CD8 leading sequence, a CD8 hinge, and transmembrane sequence, as well as the intracellular signaling domain of 4-1BB, CD28, and CD3ζ in tandem, were engineered into third generation HER2-, MSLN-, and EpCAM-CAR T cells. The sequences, except for scFv, have been previously reported (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The full-length nucleotide sequence was synthesized by Sangon Biotech (Shanghai, China), and was subsequently inserted into a CAR lentiviral expression vector [pCDH-EF1-MCS-EF1-puro] at two specific restriction enzyme sites (EcoR1 and Not1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of HER2-targeted CAR-T cells\u003c/h2\u003e \u003cp\u003eThe pCDH-CMV-MCS-EF1-puro lentivirus system was used to generate a virus against HER2-targeted CAR. HEK293T cells were co-transfected with a HER2-targeting CAR vector, or control vector, with PLP1, PLP2, and PLP-VSVG, at a ratio of 23.1:16.5:16.5:9.9, polyethyleneimine (Polysciences, Warrington, PA, USA). Six hours after transfection, cells were re-fed with DMEM containing 20% FBS. After transfection, cell supernatant was collected at 48 h and 72 h, centrifuged at 4000 rpm for 5 min to remove cell debris, then filtered through a 0.45 \u0026micro;m filter. To concentrate the virus, 1: 4 PEG8000 (Sigma, Merk, Shanghai, China) was added and mixed every 30 minutes four times. After that, the virus was placed overnight at 4\u0026deg;C, then centrifuged at 4000 g for 30 min. The supernatant was removed, and lentivirus particles were resuspended in PBS and stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eHuman peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with a Ficoll kit (GE, Shanghai, China) and subsequently activated with anti-human CD3 (100 ng/mL; T\u0026amp;L Biotechnology) and anti-human CD28 (100 ng/mL; T\u0026amp;L Biotechnology). Recombinant human IL-2 (30 ng/mL; Novoprotein) and 1% penicillin\u0026ndash;streptomycin (Gibco, Life Technologies, Shanghai, China) were added to X-VIVO 15 medium (Lonza, USA) for T cell proliferation. After activation for 24 h, T cells were transduced with HER2-targeted CAR lentiviral particles, selected with puromycin, and collected for subsequent \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments after 12\u0026ndash;14 days. Non-transduced T cells were used as a control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and culture conditions\u003c/h2\u003e \u003cp\u003eHOS, U118MG, U251, U87MG, HepG2, MKN-45, and SK-OV-3 cells were obtained from the American Type Culture Collection (ATCC, USA). HOS and SK-OV-3 cells were maintained in RPMI 1640 medium (Gibco, USA), and 293T, U118MG, U251, U87MG, HepG2, and MKN-45 cells were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco, USA). All cells were cultured in a medium supplemented with 10% FBS (BI, China), penicillin (60 \u0026micro;g/mL), and streptomycin (100 \u0026micro;g/mL) (Sangon, China) and maintained in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTissues of human glioblastoma were obtained from the Second Hospital of Dalian Medical University. Surgical glioblastoma tissues were fixed in 4% paraformaldehyde and embedded into paraffin. 4 \u0026micro;m thickness sections were deparaffinized in xylene and rehydrated with 100, 90, 80, and 70% ethanol to PBS. HER2 antibody (Cell Signaling Technology, USA) was utilized for immunostaining at room temperature (RT) for 2 hrs. After the slides were incubated with the HRP-labeled secondary Abs at RT for 1 h, 3,3' -diaminobenzidine (DAB) was added for coloration. The intensity of staining was analyzed by integrated optical density using Image-Pro R Plus software (version 6.0; Media Cybernetics, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry and antibodies\u003c/h2\u003e \u003cp\u003eCells were incubated with antiCD16/CD32 (2.4G2) mAb to block Fcγ receptors. Recombinant anti-HER2 FITC-conjugated antibody and recombinant anti-EpCAM FITC-conjugated antibody (Sino Biological Inc., Beijing, China) were used to detect the expression of HER2 and EpCAM protein. Recombinant anti-mesothelin FITC antibody (Abcam, Cambridge, UK) was used to detect the expression of mesothelin. Cells were stained with HER2, EpCAM, or mesothelin antibody for 1h on ice. The expression of CAR on CAR-T cells was detected using biotinylated human HER2/MSLN/EpCAM (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA).\u003c/p\u003e \u003cp\u003eAnti-mouse CD3 (17A2, BioLegend, USA) was used to detect the presence of CD3\u003csup\u003e+\u003c/sup\u003e T cells in mouse peripheral blood. A FACS-Calibur (Becton Dickinson, USA) was used to perform flow cytometry according to prior guidelines (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and FlowJo software (Tree Star) was utilized to analyze the data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity assays\u003c/h2\u003e \u003cp\u003eTumor cells were regarded as target cells (T) and suspended at a density (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ mL). Then, 0.1 mL cell suspension was transferred into a 96-well E-plate (ACEA Biosciences, Menlo Park, CA, USA) and cultured for 20 h. After that, HER2-targeting CAR-T cells (HER2-CAR-T) and untransfected T cells (NC-T) were regarded as effector cells (E) and added into each well separately at different E: T ratios (E: T of 5:1, or 2.5:1). The co-cultures were further cultured for the indicated times. RTCA software (xCELLigence RTCASP, ACEA, Los Angeles, CA, USA) was used to measure the viability of target cells in real time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of cytokine secretion\u003c/h2\u003e \u003cp\u003eHER2-CAR-T cells and NC-T cells were co-cultured with tumor cells for 24 h in a 96-well plate without any cytokines added. Enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, Grand Island, NY, USA) for specific cytokines (IFN-γ, TNF-α, GM-CSF, IL-6, and IL-8) were used to detect the level of cytokine production in the supernatant and assess the cell-killing efficacy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTumor models and treatment\u003c/h2\u003e \u003cp\u003e6- to 8-week-old NODPrkdcem26IL2rgem26/Nju (NCG) mice were obtained from NBRI (Nanjing Biomedical Research Institute of Nanjing University and Nanjing Galaxy Biopharma, Nanjing, China). Mice were maintained at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with free water and food intake, and illuminated for 12 hrs (08:00 to 20:00) in the specific pathogen-free (SPF) laboratory animal facility of Dalian Medical University (Dalian, China).\u003c/p\u003e \u003cp\u003eFor CDX (cell-derived xenograft, CDX) mouse models, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e U118MG tumor cells in 100 \u0026micro;L of PBS were injected subcutaneously (\u003cem\u003es.c.\u003c/em\u003e) into the axilla of NCG mice. Tumor size was measured every 4 days. Mice were divided into 3 groups with 6 mice per group until the tumor volume reached 50\u0026ndash;100 mm\u003csup\u003e3\u003c/sup\u003e. Tumor volumes were calculated according to the following formula: tumor volume = (length) \u0026times; (width)\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.5, in which the length represented the longer dimension and tumor weights were recorded. Mice were monitored according to the Institutional Animal Care and Use Committee (IACUC) animal facilities rules and regulations. Situations when the experiment needed to be paused immediately were listed, as previously reported (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e tumor killing, HER2-CAR-Ts were administered by two methods, peritumoral injection (\u003cem\u003ep.v.\u003c/em\u003e) and intravenous injection (\u003cem\u003ei.v.\u003c/em\u003e). Each U118MG-CDX mouse model was injected with 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e HER2-CAR-T cells in 200 \u0026micro;L PBS. Non-injected mice (non-transduced-T cells, named NC-T) were regarded as control groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three experiments. Two-tailed Student\u0026rsquo;s t-tests were performed to compare the statistical differences between groups, using GraphPad Prism software version 9. A p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExpression of HER2 in glioblastoma samples and tumor cell lines\u003c/h2\u003e \u003cp\u003eTo understand the relationship between HER2 expression and glioblastoma, immunohistochemical analysis of glioma surgical sections was performed with HER2 antibody. Representative immunohistochemical results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. HER2 was highly expressed in the glioma cells. To further determine whether HER2 could be a therapeutic target for tumor therapy, HER2-positive cancer cells were selected by flow cytometry. HER2 had high expression in a series of glioblastoma cells, U118MG, U251, and U87MG cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, expression of HER2 in other types of tumor cells was also detected for subsequent research. Hepatocellular carcinoma cells (HepG2), human gastric carcinoma cells (MKN-45), and cells (SK-OV-3) all had high HER2 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Human osteosarcoma cells (HOS) cells were used as negative controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Collectively, these data suggested a widespread expression of HER2 on different tumor cell types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of third-generation CART cells targeting HER2\u003c/h2\u003e \u003cp\u003eGiven the importance of HER2 in glioblastoma and also other tumors, we generated a lentiviral expression plasmid of CAR targeting human HER2 by genetic engineering. HER2-CARs include three main parts: an extracellular antigen recognition domain of the single-chain Fragment variant (scFv) derived from an anti-HER2 antibody, a CD8 transmembrane domain, and an intracellular T cell activation domain of CD3ζ. Another two vital targets, EpCAM and MSLN were also constructed in the same way for further research. Costimulatory domains included both CD28 and 4-1BB to construct CD3ζ-CD28-41BB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Lentivirus was produced by co-transfecting 293T cells with the HER2 CAR plasmid, PLP1, PLP2 and PLP-VSVG. HER2, EpCAM, and MSLN CAR-T cells were prepared by lentiviral infection using PBMCs. The efficiency of infection was evaluated by flow cytometry using the anti-HER2, anti-EpCAM, and anti-MSLN antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity of HER2 CART cells in glioblastoma\u003c/h2\u003e \u003cp\u003eTo evaluate the cytotoxicity and specificity of CAR-T cells, based on high expression of HER2, but not other targets, different targeting CAR-T cells were co-cultured with U118MG cells. Anti-HER2 CAR-T, anti-EpCAM CAR-T, and anti-MSLN CAR-T cells were cultured for 10 days and then independently co-cultured with U118MG cells at an effector-to-target (E: T) ratio of 2.5:1 and 5:1, with the NC-T (non-transduced T cells) group used as the control group. Real-time cytotoxicity assays (RTCA) provide automated real-time data acquisition continuously to monitor CAR-T cell-mediated killing of cancer cells and gain deeper insights into the specificity, potency, persistence, and efficiency of CAR-T cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, compared with anti-EpCAM CAR-T and anti-MSLN CAR-T, the efficiency of HER2-CAR-T presented a stronger cell-killing ability on U118MG cells. Moreover, the cell-killing effect at an E: T ratio of 5:1 was greater than that at a 2.5:1 E: T ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that HER2-CAR-T cells had a dose-dependent effect on tumor cell killing. The cell killing rate was also analyzed and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is shown that T cell-derived TNF-α and IFN-γ are required for T-cell mediated killing of established tumors (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Next, we compared the cytokine release mediated by HER2-CAR-T and NC-T against HER2-positive glioblastoma cell lines. IFN-γ and TNF-α released in the supernatant were all present at high levels in HER2-CAR-T-targeted U118MG, U251, and U87-MG cells, compared with the corresponding NC-T targeted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results demonstrate that the effect of HER2-CAR-T cells on tumors is target-specific.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnti-tumor efficacy of HER2 CAR-T cells in vivo\u003c/h2\u003e \u003cp\u003eSince HER2 CAR-T cells are cytotoxic toward HER2-positive tumor cells \u003cem\u003ein vitro\u003c/em\u003e, we speculated that HER2-CAR-T cells could also play a prominent part in tumor cell killing in vivo. We first evaluated the influence of HER2-CAR-T cells on tumor cell killing in tumor-bearing mice via two means of injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). NCG mice, inoculated subcutaneously with U118MG cells, were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;8): an NC-T control group (NC-T), a peritumoral injection group (HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e), and an intravenous injection group (HER2-CAR-T-\u003cem\u003ei.v.\u003c/em\u003e). Once tumors reached 50 mm\u003csup\u003e3\u003c/sup\u003e, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e effector cells were administered in different ways. Twenty days after effector cell injection, compared with the NC-T group, a significant tumor suppressive effect was observed both in the HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e group and HER2-CAR-T-\u003cem\u003ei.v.\u003c/em\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Day 44 after CAR-T cell implantation, NC-T mice were euthanized due to the enormous volume of tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e group and HER2-CAR-T-\u003cem\u003ei.v.\u003c/em\u003e group were still fed to observe the influence of different injection modes of CAR-T cells on tumor therapy. On day 44, peripheral blood was collected from each mouse and the proportion of CD3\u003csup\u003e+\u003c/sup\u003e T cells was measured. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, the proportion of CD3\u003csup\u003e+\u003c/sup\u003e T cells in peripheral blood was still at higher levels in the HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e group and HER2 CAR-T-\u003cem\u003ei.v.\u003c/em\u003e group, while in the NC-T group was depleted. To further investigate CAR-T cell efficacy, tumor tissue was obtained surgically, and CAR-T cells were measured via flow cytometry. Notably, the proportions of CAR-T cells were kept at high levels in the HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e group and HER2-CAR-T-\u003cem\u003ei.v.\u003c/em\u003e group compared with the NC-T group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Interestingly, the HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e group displayed a much higher proportion of tumor-infiltrating HER2-CAR-T cells in comparison with the HER2 CAR-T-\u003cem\u003ei.v.\u003c/em\u003e group, implying a better efficiency of peritumoral administration towards glioma (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the effect of CAR-T treatment on glioma, a long-term increase in tumor volume was measured under CAR-T therapy by \u003cem\u003ep.v.\u003c/em\u003e and \u003cem\u003ei.v.\u003c/em\u003e Tumor volume was smaller in the HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e group, but was not statistically significant until day 72 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We subsequently measured GM-CSF (granulocyte-macrophage colony-stimulating factor) and IFN-γ in the serum of HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e and HER2-CAR-T-\u003cem\u003ei.v.\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Elevated GM-CSF and IFN-γ were found in the HER2-CAR-T mice, especially in HER2-CAR-T-\u003cem\u003ep.v.\u003c/em\u003e mice, while IL-8 and IL-6 exhibited decreased expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Taken together, our results suggest that CAR-T therapy by \u003cem\u003ep.v.\u003c/em\u003e exerts a stronger therapeutic effect on gliomata compared with \u003cem\u003ei.v.\u003c/em\u003e administration. Based on these results, we propose that HER2-CAR-T therapy could be considered as a rational immunotherapeutic strategy, and peritumoral administration could be an innovative and locoregional approach for GBM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study, we found an anti-tumor activity of HER2-CAR-T cells against HER2-positive GBM cells, as well as other HER2-positive tumor cell types. However, high tumor heterogeneity, local physical barriers, hostile tumor microenvironment, and antigen escape still make CAR-T therapy a challenging treatment (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The survival of GBM cells in the harsh local environment of the brain and the elimination of multiple types of tumor antigens also pose unique difficulties that need to be solved. Tumor-associated antigens (TAA) are the main targets for CAR-T-engineered therapy (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The critical therapeutic barrier is the diverse expression of TAAs of GBM cells. Moreover, apart from HER2, various expression levels of other antigens in GBM also impair the function of HER2-CAR-T because the diversity of GBM cell antigens makes it impossible to identify HER2 antigen, thereby affecting the efficiency of tumor treatment. Till today, there has been a series of CAR-T therapeutic targets, towards GBM, involving EGFRvIII, IL13Ra2, HER2, B7-H3, CD70, GD2, MMP2, and NKG2D (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To explore the targeting of multiple TAAs on GBM by identified CAR-T cells, including the co-expression of several CARs on a single T cell, and expression of a chimeric receptor including two or more antigen recognition domains, which in turn leads to the identification of multiple antigens through individual receptors (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) are both more effective treatment for GMB to conquer the tumor heterogeneity. Combining CAR-T treatment with the other immunotherapies was also a meaningful strategy for tumor therapy. In hematological malignancy, a combination of PD-1 blockade and CD19 CAR-T cell therapy in B-ALL patients improved outcomes and improved CAR-T cell persistence (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). A recent study has shown that third-generation HER2-specific CAR-T cells can efficiently eliminate GBM cells \u003cem\u003ein vitro\u003c/em\u003e and that the activity of the administered CAR-T cells is increased by their combination with PD-1 blockade (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBefore CAR-T cells can get to work, they must bypass the blood-brain barrier (BBB), a non-fenestrated physical barrier comprised of specialized capillary endothelial cells interconnected by multi-protein tight junctions (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Unlike hematological malignancies, the peripheral blood is not the compartment of therapeutic action, and the effective CAR-T cell dose and frequency/schedule of administration are elusive in GBM. In addition to the above, the major concern when targeting HER2 is potential side effects due to HER2 expression in various normal tissues, especially in vital organs, although this has rarely been an issue with HER2-specific CAR-T cell administration in humans thus far. During CAR-T treatment, a high accumulation of CAR-T cells occurs in normal lung and abdominal/mediastinal lymph nodes, on which HER2 is expressed, although at a low level (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), which could reduce the efficiency of tumor treatment. Higher infusion of a large number of CAR-T cells can trigger the release of life-threatening supraphysiological levels of pro-inflammatory cytokines which could cause serious side effects and disorders. Taken together, the innovation of CAR-T cell delivery is extremely critical for GBM therapy. We establish a peritumoral injection strategy of CAR-T in a CDX mouse model. Compared with the traditional approach using intravenous injection, we show peritumoral injection of CAR-T cells more efficiently primes locoregional immunity for GBM therapy. Furthermore, CAR-T therapy via peritumoral injection can be combined with novel strategies to enhance CAR-T cell cytotoxic ability to GBM.\u003c/p\u003e \u003cp\u003eCurrently, CAR-T cells can be classified into four different generations, with next or fifth-generation CARs currently under active development (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Here, engineered third-generation CAR-T cells were utilized, which combined 4-1BB and CD28 signaling domains to provide superior activation and proliferation capacity compared with second-generation CAR-T cells. However, overstimulation of T-cell activity by two costimulatory molecules can induce sharp increases in cytokine secretion, leading to cytokine release syndrome (CRS). Recently, the original CD3ζ has been replaced with three peptide chains of CD3, γ, δ, and ε which can be used to solve the problem of T cell depletion, exhaustion, and CRS during CAR-T treatment (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Among all CAR activations, BB-ζ secreted the highest level of cytokines, and a large portion of cytokines was related to CRS, suggesting that the use of CAR with other three peptide chains could reduce the occurrence of CRS and enhance the safety of CAR-T therapy (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). A CAR that uses one of the signaling domains of another peptide chain rather than the ζ chain could mitigate or prevent the shortcomings of existing CAR-T cell therapies (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Further research will focus on the therapeutic efficiency of the other peptide chains of CD3. Fourth-generation CAR-T cells incorporate cytokines or co-stimulatory ligands to further enhance the T cell response, or suicide genes to cause the CAR-T cells to self-destruct if needed. Regulatory elements of suicide genes could increase the safety and targeting of CAR-T therapy. Fourth-generation CAR-T cells, redirected for universal cytokine killing, could secrete specific cytokines (currently mainly IL-12) in the tumor region, thereby modifying the tumor microenvironment, and recruiting and activating other immune cells for an immune response. Fifth-generation CAR-T technology will break through individual limitations to be universal and produced on a large-scale, and enable treatment among different individuals. The development of new CAR-T therapies should therefore consider strategies that can achieve a more balanced immune response.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no commercial or financial conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eZhang NZ designed the manuscript; Li XY wrote the manuscript; Zhao LF prepared the figures; Li WZ revised the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was Supported by the National Natural Science Foundation of China, No. 32171279; Natural Science Foundation of Liaoning Province, No. 2022-BS-254.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHuang B, Li X, Li Y, Zhang J, Zong Z, Zhang H. Current Immunotherapies for Glioblastoma Multiforme. Frontiers in immunology. 2020;11:603911.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYalamarty SSK, Filipczak N, Li X, Subhan MA, Parveen F, Ataide JA, et al. Mechanisms of Resistance and Current Treatment Options for Glioblastoma Multiforme (GBM). Cancers. 2023;15(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePardridge WM. Drug transport across the blood-brain barrier. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 2012;32(11):1959\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood cancer journal. 2021;11(4):69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorchakov AA, Kulemzin SV, Kochneva GV, Taranin AV. Challenges and Prospects of Chimeric Antigen Receptor T-cell Therapy for Metastatic Prostate Cancer. European urology. 2020;77(3):299\u0026ndash;308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Z, Chen Y, Francisco NM, Zhang Y, Wu M. The application of CAR-T cell therapy in hematological malignancies: advantages and challenges. Acta pharmaceutica Sinica B. 2018;8(4):539\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandoni E, Savoldo B. Treating hematological malignancies with cell therapy: where are we now? Expert opinion on biological therapy. 2018;18(1):65\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavani M, Oluwole O, Dholaria B. New targets for CAR T therapy in hematologic malignancies. Best practice \u0026amp; research Clinical haematology. 2021;34(3):101277.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei J, Han X, Bo J, Han W. Target selection for CAR-T therapy. Journal of hematology \u0026amp; oncology. 2019;12(1):62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarofi F, Motavalli R, Safonov VA, Thangavelu L, Yumashev AV, Alexander M, et al. CAR T cells in solid tumors: challenges and opportunities. Stem cell research \u0026amp; therapy. 2021;12(1):81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nature reviews Drug discovery. 2021;20(7):531\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarson RC, Kann MC, Bailey SR, Haradhvala NJ, Llopis PM, Bouffard AA, et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature. 2022;604(7906):563\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen C, Jing W, Chen Y, Wang G, Abdalla M, Gao L, et al. Intracavity generation of glioma stem cell-specific CAR macrophages primes locoregional immunity for postoperative glioblastoma therapy. Science translational medicine. 2022;14(656):eabn1128.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Li Y, Gu L, Chen R, Zhu H, Zhang X, et al. Gene Targets of CAR-T Cell Therapy for Glioblastoma. Cancers. 2023;15(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMineo JF, Bordron A, Baroncini M, Maurage CA, Ramirez C, Siminski RM, et al. Low HER2-expressing glioblastomas are more often secondary to anaplastic transformation of low-grade glioma. Journal of neuro-oncology. 2007;85(3):281\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHynes NE, MacDonald G. ErbB receptors and signaling pathways in cancer. Current opinion in cell biology. 2009;21(2):177\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed N, Salsman VS, Kew Y, Shaffer D, Powell S, Zhang YJ, et al. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors. Clinical cancer research: an official journal of the American Association for Cancer Research. 2010;16(2):474\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePress MF, Cordon-Cardo C, Slamon DJ. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene. 1990;5(7):953\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Liu G, Liu J, Yang M, Fu J, Liu G, et al. The antitumor capacity of mesothelin-CAR-T cells in targeting solid tumors in mice. Molecular therapy oncolytics. 2021;20:556\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCossarizza A, Chang HD, Radbruch A, Acs A, Adam D, Adam-Klages S, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). European journal of immunology. 2019;49(10):1457\u0026ndash;973.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Zhang Z, Liu G, Li D, Gu Z, Zhang L, et al. B7-H3 targeted CAR-T cells show highly efficient anti-tumor function against osteosarcoma both in vitro and in vivo. BMC cancer. 2022;22(1):1124.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang B, Karrison T, Rowley DA, Schreiber H. IFN-gamma- and TNF-dependent bystander eradication of antigen-loss variants in established mouse cancers. The Journal of clinical investigation. 2008;118(4):1398\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKankeu Fonkoua LA, Sirpilla O, Sakemura R, Siegler EL, Kenderian SS. CAR T cell therapy and the tumor microenvironment: Current challenges and opportunities. Molecular therapy oncolytics. 2022;25:69\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu D. Cancer biomarkers for targeted therapy. Biomarker research. 2019;7:25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan JF, Khan AS, Brentjens RJ. Application of CAR T cells for the treatment of solid tumors. Progress in molecular biology and translational science. 2019;164:293\u0026ndash;327.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong W, Zhang M. Use of CAR-T cell therapy, PD-1 blockade, and their combination for the treatment of hematological malignancies. Clinical immunology (Orlando, Fla). 2020;214:108382.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaggs L, Cattaneo G, Dal AE, Moghaddam AS, Ferrone S. CAR T Cell-Based Immunotherapy for the Treatment of Glioblastoma. Frontiers in neuroscience. 2021;15:662064.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBallabh P, Braun A, Nedergaard M. The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiology of disease. 2004;16(1):1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelasco C\u0026aacute;rdenas RM, Brandl SM, Mel\u0026eacute;ndez AV, Schlaak AE, Buschky A, Peters T, et al. Harnessing CD3 diversity to optimize CAR T cells. Nature immunology. 2023;24(12):2135\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3926646/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3926646/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. Current treatment options for GBM include surgical resection, radiation, and chemotherapy, which predominantly only slow cancer growth and reduce symptoms, resulting in a 5-year survival rate of no more than 10%. Chimeric antigen receptor (CAR)-T cell therapy is a new class of cellular immunotherapies that has made great progress in the treatment of malignant tumors. Human epidermal growth factor receptor 2 (HER2) is over-expressed in GBM, and may provide a potential therapeutic target for GBM treatment. In this study, we constructed third-generation CAR-T cells targeting the HER2 antigen in GBM. HER2-CAR-T cells showed effective antitumor abilities both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Furthermore, HER2-specific CAR-T cells exhibited strong cytotoxicity and cytokine-secreting ability against GBM cells \u003cem\u003ein vitro\u003c/em\u003e. Anti-HER2 CAR-T cells also exerted increasing cytotoxicity from low to high effector-to-target (E: T) ratios. Importantly, anti-HER2 CAR-T cells delivered by peritumoral injection successfully stunted tumor progression \u003cem\u003ein vivo\u003c/em\u003e. Moreover, peritumoral intravenous administration of anti-HER2 CAR-T exhibited a therapeutic improvement against GBM cells compared with intravenous administration. In conclusion, our studies show that HER2 CAR-T cells represent an emerging immunotherapy for the treatment of GBM.\u003c/p\u003e","manuscriptTitle":"HER2-targeting CAR-T cells show highly efficient anti-tumor activity against glioblastoma both in vitro and in vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-13 19:42:30","doi":"10.21203/rs.3.rs-3926646/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-03-04T09:24:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-02-25T05:23:37+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-02-25T03:55:10+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-02-21T06:11:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-16T07:40:58+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-11T00:18:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-10T11:54:06+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-02-10T10:40:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-06T16:40:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genes \u0026 Immunity","date":"2024-02-06T01:11:35+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-02-05T11:55:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-04T07:15:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genes-and-immunity","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"genes","sideBox":"Learn more about [Genes \u0026 Immunity](http://www.nature.com/gene/)","snPcode":"41435","submissionUrl":"https://mts-gene.nature.com/cgi-bin/main.plex","title":"Genes \u0026 Immunity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"02531e74-db37-46c9-91c7-99008d1f0be9","owner":[],"postedDate":"February 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28611488,"name":"Biological sciences/Immunology/Immunotherapy/Immunosuppression"},{"id":28611489,"name":"Biological sciences/Immunology/Tumour immunology/Immunosurveillance/Immunoediting"}],"tags":[],"updatedAt":"2024-05-04T00:46:14+00:00","versionOfRecord":{"articleIdentity":"rs-3926646","link":"https://doi.org/10.1038/s41435-024-00275-6","journal":{"identity":"genes-and-immunity","isVorOnly":false,"title":"Genes \u0026 Immunity"},"publishedOn":"2024-05-03 04:00:00","publishedOnDateReadable":"May 3rd, 2024"},"versionCreatedAt":"2024-02-13 19:42:30","video":"","vorDoi":"10.1038/s41435-024-00275-6","vorDoiUrl":"https://doi.org/10.1038/s41435-024-00275-6","workflowStages":[]},"version":"v1","identity":"rs-3926646","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3926646","identity":"rs-3926646","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}