Development of a new EGFR-VHH-CAR T-cell therapy for treatment of esophageal squamous cell carcinoma

Development of a new EGFR-VHH-CAR T-cell therapy for treatment of esophageal squamous cell carcinoma | 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 Development of a new EGFR-VHH-CAR T-cell therapy for treatment of esophageal squamous cell carcinoma Chenglin Zhang, Yanyan Liu, danya zhou, haoran guo, ying peng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4485969/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Esophageal squamous cell carcinoma (ESCC), a primary form of esophageal cancer, is characterized by poor outcomes and limited treatment options. Targeting EGFR with chimeric antigen receptor-modified T cells (EGFR CAR-T) has emerged as a promising therapeutic approach for ESCC. Utilizing nanobodies enhances the specificity of antigen recognition and has become a popular method in CAR-T cell therapy. In this study, we introduced an EGFR-specific nanobody, EGFR-VHH-7D12, into the receptor binding domain of EGFR CAR-T cells and incorporated human interleukin 21 (hIL-21) to boost the efficacy of these cells. Our findings show that EGFR-VHH-7D12-equipped CAR-T cells can accurately target and eliminate EGFR-positive esophageal cancer cells both in vitro and in animal models. The addition of hIL-21 not only increased the proliferation of CAR-T cells but also led to a higher formation of memory T cell subsets in vitro. Furthermore, the presence of hIL-21 in these CAR-T cells resulted in increased expression of IFN-gamma when cultured with various human esophageal cancer cell lines (KYSE-30, KYSE-150, and KYSE-510) in vitro. Notably, CAR-T cells expressing both EGFR-VHH-7D12 and hIL-21 showed superior anti-tumor activity in a KYSE-150 xenograft mouse model. Our results indicate that the combined expression of hIL-21 in 7D12-CAR-T cells significantly enhances their anti-tumor capabilities, making them a highly promising option for ESCC treatment. Biological sciences/Cancer Biological sciences/Immunology ESCC EGFR Nanobody CAR-T cells human IL-21 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Esophageal cancer among the top 10 most prevalent malignancies globally, characterized by high morbidity and mortality rates [1]. Esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) are two primary subtypes of esophageal cancer. Notably, China harbors over half of the global esophageal cancer patient population, with ESCC constituting more than 90% of these cases[2]. ESCC is particularly aggressive, exhibiting rapid progression, a high recurrence rate, and a dismal 5-year survival rates below 30%, underscoring the urgency for advanced clinical research[3]. Conventional systemic chemotherapy, typically involving cisplatin and 5-fluorouracil (5-FU) (CF), remains the standard treatment for ESCC [4, 5]. However, the limited anti-tumor efficacy and poor prognosis have necessitated the exploration of more effective therapeutic strategies. EGFR, a receptor tyrosine kinase abundantly expressed on ESCC cells and other epithelial-derived tumors with poor prognosis, serves as a target for CAR-T cell therapy[6]. Its overexpression in ESCC patients, contrasted with low expression in normal tissues, positions EGFR as a prime therapeutic target[7]. Previous clinical trials involving EGFR-targeted CAR-T cell therapy in various cancers, including triple-negative breast cancer, head and neck cancer, glioblastoma, and non-small cell lung cancer, have shown promising outcomes[8–11]. Chimeric antigen receptor-modified T (CAR-T) cells are engineered by introducing a single-chain fragment variable (ScFv) and an intracellular co-stimulatory domain, such as 4-1BB and CD3ζ to activate and enhance the cytotoxicity of T cells[12]. These modified T cells can specifically target and eliminate tumor cells without the need for major histocompatibility complex (MHC) presentation[13]. However, challenges with ScFv, including improper folding and aggregation, have limited the efficacy of CAR-T cells, prompting the search for alternatives[14]. The variable domain of heavy chain of heavy-chain antibodies (VHHs), also named nanobodies, emerges as a promising substitute for ScFv due to their small size (15kDa), which facilitates binding to complex, and their reduced immunogenicity thanks to humanization modification[15]. VHH-based CAR-T therapy, particularly targeting B cell maturation antigen (BCMA), has been FDA-approved for its potent tumor-suppressive function in multiple myeloma (MM), achieving an 88% overall response (OR) and 68% complete response (CR)[16]. In this research, we introduced the EGFR-VHH-7D12, a nanobody targeting the third domain of EGFR with high binding affinity, offering potential application in the for EGFR-targeted VHH-based CAR-T therapy[17]. Furthermore, we investigated human interleukin 21, a multifunctional cytokine from the common g chain family, known for its role in promoting CD8 + killer T cell proliferation and enhancing interferon-gamma (IFN‐γ) secretion[18, 19]. Unlike lIL-2, lIL-7, and lIL-15, IL-21 activates signal transducer and activators of transcription 1(STAT1) and 3 (STAT3), but not STAT5A or STAT5B, indicating its unique influence on T cell anti-tumor functions[20]. Here, we developed two novel nanobody-based CAR-T cells: 7D12-CAR-T cells (7D12-CAR) and 7D12-CAR-T cells expressing human IL-21(7D12-CAR-hIL-21). Both constructs specifically target EGFR-positive ESCC cell lines, with the latter demonstrating enhanced persistence in expressing human IL-21 and superior anti-tumor efficacy in vitro and in vivo. Results EGFR was Upregulated in ESCC with Predicted Poor Prognosis The aberrantly activated tyrosine kinase pathway, closely linked to EGFR, plays a pivotal role in promoting anti-apoptosis and carcinogenesis in normal epithelial cells. Notably, EGFR's aberrant expression is a common feature in epithelial tissue malignancies, correlating strongly with poor prognosis, particularly in esophageal squamous cell carcinoma (ESCC)[21]. Given the variable EGFR expression across different cell lines, we embarked on a comparative analysis of EGFR levels in various KYSE cell lines (KYSE-30, KYSE-70, KYSE-140, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, KYSE-510) and normal human esophageal epithelial cells (HET-1A) using flow cytometry (Fig. 1 A). Our findings revealed a prevalent overexpression of EGFR in KYSE cell lines, albeit with varying intensities, in contrast to its lower expression in normal human esophageal epithelial cells (Figs. 1 B and 1 C). Subsequent mRNA expression analysis of EGFR (Fig. 1 D) corroborated the flow cytometry outcomes, particularly for KYSE-30, KYSE-150, and KYSE-510, which demonstrated high, medium, and low levels of EGFR expression, respectively. for further investigate the efficacy of our CAR-T cells, we selected these three KYSE cell lines (KYSE-30, KYSE-150, and KYSE-510) based on their distinct EGFR expression levels for subsequent experiments. Generation of EGFR- specific VHH-7D12 nanobody CAR-T cells The nanobody EGFR-VHH-7D12, recognized for its ability to specifically target the membrane surface protein EGFR, has been increasingly utilized in immunotherapy applications over the past few years[17]. IL-21, a potent immunoregulatory cytokine, is instrumental in the immune response, enhancing the activation and proliferation of T cells and natural killer cells [22, 23]. To assess the specificity of EGFR-VHH-7D12, we designed an extracellular segment of EGFR protein EGFR Met1−Ser645 -mouse IgG Fc fusion protein (eEGFR-mIgG-Fc). The architecture of this fusion protein is depicted in Fig. 2 A (top panel). The EGFR-VHH-7D12 was employed as an alternative to ScFv to construct the 7D12-CAR, incorporating EGFR-VHH-7D12, CD8 hinge and transmembrane domains, 4-1BB, and CD3zeta domains. Subsequently, the 7D12-CAR-hIL-21 variant was engineered by appending a 2A peptide and human IL-21 subsequent to the CD3ζ intracellular signaling domain (Fig. 2 A, middle and bottom panels). For the production of the EGFR Met1−Ser645 -mIgG-Fc protein, 293E cells were utilized, followed by concentration of the detection protein using a 50KD ultrafiltration tube at 1500 xg. The medium, EGFR Met1−Ser645 -mIgG-Fc infusion protein, and a 10-fold concentrated version of the EGFR Met1−Ser645 -mIgG-Fc infusion protein were subsequently analyzed via Western blot (Fig. 2 B and Supplemental Fig. 1). To assess the expression of EGFR-VHH-7D12, we utilized the commercial anti-VHH cocktail-FITC antibody, targeting the conserved sequence of VHH, to investigated the stability of EGFR-VHH-7D12 expression across T cells from three different donors. Flow cytometry analysis revealed that T cells, at a multiplicity of infection (MOI) of 5, exhibited EGFR-VHH-7D12 expression on the surface of both 7D12-CAR-T and 7D12-CAR-hIL-21-T cells, but not on UTD T cells (Fig. 2 C and Supplement Fig. 2 ). Further, we investigated the recognition ability of EGFR-VHH-7D12 by utilizing the concentrated EGFR Met1−Ser645 -mIgG-Fc infusion protein as a primary antibody in both untransduced (UTD) T cells and 7D12-CAR-T cells. For comparison, commercial anti-VHH cocktail-FITC antibody served as the positive control. The combination of EGFR Met1−Ser645 -mIgG-Fc infusion protein with anti-mouse IgG-Fc-FITC enabled the detection of EGFR-VHH-7D12 expression in 7D12-CAR-T cells. Notably, the recognition rate was slightly better compared to the commercial antibody (86.3% vs. 84.4%) (Fig. 2 D). Neither antibody yielded positive results in UTD T cells (Supplement Fig. 3 ), indicating the specificity of EGFR-VHH-7D12 for EGFR protein and validating our EGFR Met1−Ser645 -mIgG-Fc infusion protein's ability to specifically target EGFR on the cell surface. These findings collectively demonstrate the specific targeting capability of EGFR-VHH-7D12 towards EGFR protein and the successful generation of EGFR-specific VHH-7D12 nanobody CAR-T cells. Influence of Human IL-21 on CAR-T Cell Subpopulations IL-21 has been documented to significantly affect various T cell cultures. To determine the impact of IL-21 in CAR-T cells proliferation and differentiation, we first detected the expression of IL-21 in 7D12-CAR-hIL-21-T cells. The secretion of hIL-21 by 7D12-CAR-hIL-21-T cells from all three donors was quantified, exceeding 2000 pg/ml at an MOI of 5 (Fig. 3 A). The influence of hIL-21 on promoting CAR-T cell proliferation in vitro was subsequently examined. Results indicated that 7D12-CAR-hIL-21-T cells exhibited enhanced proliferative capabilities compared to both UTD T cells and 7D12-CAR-T cells (Fig. 3 B). Our study further elucidates its impact on CAR-T cell subpopulations. After 11 days of in vitro culture, 7D12-CAR-hIL-21-T cells exhibited a decrease in CD4 + T cell proportion (50.27%±0.484% vs. 63.40%±0.866%, P < 0.001) and an increase in CD8 + T cells (40.73%±0.491% vs. 28.07%±0.066%, P < 0.001) compared to 7D12-CAR-T cells (Fig. 3 C-E). This shift suggests IL-21's role in modulating the CD4+/CD8 + T cell ratio within the CAR-T cell milieu. Moreover, we investigated the memory T cell subsets between 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells. The latter group demonstrated a higher expression of CD3+/CD4+/CD45RO + CD62L + central memory T cells (83.67%±1.898% vs. 67.8% ± 0.2517%, P < 0.01) and CD3+/CD8+/CD45RO + CD62L + central memory T cells (55.63%±0.6984% vs. 43.93%±0.4910%, P < 0.01) compared to 7D12-CAR-T cells (Figs. 3 F-H). To delve deeper into hIL-21's functional impact on CAR-T cells, we performed transcriptome analysis on both 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells after 11 days of in vitro culture. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified the MAPK signaling pathway as significantly modulated, which may enhance the proliferation of CAR-T cells (Supplement Fig. 4 A)[24]. Further analysis of genes related to the MAPK signaling pathway, with a log2 scale cutoff of 0.58, was visualized through a heatmap, delineating the gene expression changes within this pathway (Supplement Fig. 4 B). These comprehensive analyses suggest that IL-21 plays a pivotal role in enhancing the proliferation ability and skewing the CAR-T cell population towards a more cytotoxic phenotype, with increased proportions of CD8 + T cells and CD3+/CD8+/CD45RO + CD62L + central memory cytotoxic T cells. Additionally, an increase in CD3+/CD4+/CD45RO + CD62L + central memory T cells was observed and the change of MAPK signaling pathway, highlighting IL-21's broad immunomodulatory effects. In Vitro Cytotoxicity of EGFR-CAR T Cells Against ESCC Cell Lines EGFR is prevalently overexpressed in esophageal squamous cell carcinomas (ESCCs), highlighting its potential as a therapeutic target[23]. To evaluate the antitumor efficacy of 7D12-CAR-T and 7D12-CAR-hIL-21-T cells against ESCC with varying EGFR expression levels, we utilized KYSE-30, KYSE-150, KYSE-510, and an EGFR-knockout cell line as targets, as detailed by western blot (Supplemental Fig. 5A). Both CAR-T cell variants demonstrated specific and potent cytotoxicity against the KYSE-30, KYSE-150, and KYSE-510 cell lines (Figs. 4 A-C). In contrast, cytotoxic activity was negligible against the EGFR-knockout cell line, indicating the specificity of the CAR-T cells' action (Supplemental Fig. 5B). Remarkably, 7D12-CAR-hIL-21-T cells outperformed their counterparts in lysing all three ESCC cell lines, underlining the enhanced efficacy conferred by hIL-21. Furthermore, we assessed the secretion of cytotoxic cytokines, IFN-γ and TNF-α, during co-culture with tumor cells at an effector-to-target (E:T) ratio of 16:1. 7D12-CAR-hIL-21-T cells exhibited elevated levels of IFN-γ secretion compared to 7D12-CAR-T cells (Figs. 4 D-F), while TNF-α levels remained comparable across both CAR-T cell types (Figs. 4 G-I). These findings underscore the nanobody-based CAR-T cells' robust and consistent antitumor efficacy across different ESCC cell lines. Additionally, the incorporation of hIL-21 appears to augment the anti-tumor potency of CAR-T cells, potentially through the upregulation of IFN-γ expression. Efficacy of EGFR-CAR T Cells in ESCC Cell Mouse Xenograft Models This study evaluated the therapeutic potential of 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells against esophageal squamous cell carcinoma (ESCC) growth in mouse xenograft models. Non-obese diabetic/severe combined immunodeficient/γ-chain−/− (NSG) mice were subcutaneously inoculated with 5.0x10^6 KYSE-150 cells. Upon tumor volumes reaching approximately 100 mm 3 , mice were administered 5.0x10 6 CAR-T cells, 5.0x10 6 untransduced (UTD) T cells, or phosphate-buffered saline (PBS) in a 100µl volume (Fig. 5 A). Our observations revealed that both CAR-T cell variants significantly inhibited tumor proliferation and enhanced survival rates, outperforming the UTD T cells and PBS control groups. Among them, the 7D12-CAR-hIL-21-T cells displayed markedly superior tumor growth suppression compared to both the UTD T cells and 7D12-CAR-T cells (Fig. 5 B). A minor reduction in body weight was noted in the initial phase of 7D12-CAR-T cell treatment; however, no significant weight loss was observed throughout the experiment (Fig. 5 C). Cytokine analysis in peripheral blood showcased sustained hIL-21 expression exclusively in the 7D12-CAR-hIL-21-T cell treatment group, with no appreciable difference in IFN-γ levels between the two CAR-T cell groups (Fig. 5 D). Notably, the presence of hIL-21 was confirmed in the peripheral blood of the 7D12-CAR-hIL-21-T cell group (Fig. 5 E). These results underscore the effectiveness of EGFR-VHH CAR-T cell therapies in curtailing the growth of KYSE-150 subcutaneous tumors, with the 7D12-CAR-hIL-21-T cells demonstrating enhanced therapeutic efficacy. Safety Profile of EGFR-CAR T Cells in ESCC Cell Mouse Xenograft Models Given that EGFR serves as a tumor-associated antigen, evaluating the on-target, off-tumor (OTOT) risk associated with EGFR-VHH-7D12 CAR-T cell therapy is imperative[23]. Our initial investigation focused on potential tissue damage in mouse xenograft models treated with phosphate-buffered saline (PBS), 7D12-CAR-T cells, and 7D12-CAR-hIL-21-T cells. Immunohistochemical analysis across these groups revealed no significant tissue damage in critical organs, including the heart, liver, spleen, lung, and kidney (Fig. 6 A). Despite the minimal EGFR expression in these tissues, we further examined the potential infiltration of CAR-T cells into normal tissues. Genomic DNA extracted from major organs was subjected to PCR amplification using WPRE-specific primers. WPRE sequences were detected in the 7D12-CAR-T and 7D12-CAR-hIL-21-T cell treatment groups, but not in the PBS control group (Fig. 6 B). While immunohistochemistry did not indicate overt tissue damage, the presence of CAR-T cells in various tissues suggests a potential OTOT risk. Additionally, we assessed liver function by measuring levels of liver enzymes (ALT, AST, ALP) to identify any signs of liver damage. The results showed no significant differences in enzyme levels between the CAR-T cell therapy groups and the control group (Figs. 6 C-E). These findings collectively suggest that EGFR-VHH-7D12-CAR-T cell therapy does not induce significant tissue damage, despite evidence of CAR-T cell presence in non-target tissues. This comprehensive safety assessment underscores the potential of VHH-7D12-based CAR-T cell therapy for clinical application, highlighting its favorable safety profile in the context of ESCC treatment. Discussion Chimeric antigen receptor (CAR) -modified T cells have demonstrated remarkable efficacy in identifying and destroying tumor cells, particularly in hematological malignancies[25]. However, their anti-tumor capability in solid tumors, including ESCC, remains limited[26]. In our study, we developed a new nanobody-based anti-EGFR-CAR-T cells, incorporating hIL-21 to enhance their anti-tumor activity against ESCC. Our findings indicate that both 7D12-CAR-T cells, and 7D12-CAR-hIL-21-T cells can effectively inhibit tumor growth. Notably, the 7D12-CAR-hIL-21-T cells performed superior proliferation, increased memory T cells subgroups, and more potent anti-tumor effects both in vitro and in vivo. These results underscore the potential of our nanobody-based anti-EGFR-CAR-T cells, especially those expressing hIL-21, as a promise treatment for ESCC, offering a new immunotherapeutic strategy for this aggressive cancer. The Epidermal Growth Factor Receptor (EGFR), part of the epidermal growth factor receptor family, is commonly present on epithelial-derived tissue cell membranes [27, 28]. EGFR activation, through ligand binding and subsequent dimerization, initiates signaling cascades that promote cell proliferation, angiogenesis, invasion, metastasis, and apoptosis inhibition[29]. EGFR-targeted CAR-T cells have shown efficacy in various tumor types, including triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), multiple glioblastoma (GBM), and ESCC[8–11]. High expression of EGFR is associated with poor prognosis[21]. In this study, we assessed EGFR expression across nine ESCC cell lines, selecting KYSE-30, KYSE-150, and KYSE-510 to represent varying EGFR expression levels. Both 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells successfully targeted and eliminated these three tumor cell lines in vitro. In 1993, researchers identified a naturally occurring heavy-chain antibody in dromedary camels, known as heavy-chain antibodies (HCAb), which lack light chains[30]. Through bioengineering, the heavy chain variable region domain of HCAb can be cloned to create VHH antibodies, also called nanobodies[31]. Compared to scFv, nanobody-based CAR-T cells exhibit superior characteristics, such as lower immunogenicity, easier folding outside the cell membrane, reduced likelihood of abnormal aggregation, and enhanced stability[32]. Carvykti, a BCMA-targeted nanobody-based CAR-T cell, is the first FDA-approved commercial nanobody CAR-T product, signaling the strong potential of nanobodies in CAR-T cell therapy[33]. In this study, novel nanobody-based CAR-T cells targeting EGFR were developed based on the VHH sequence 7D12 (EGFR-VHH-7D12). This VHH have been applied in photodynamic therapy (PDT) by conjugated with photosensitizing chemical substance (7D12-PS)[34]. This 7D12-PS performed high anti-tumor ability without damaging normal tissues. We designed and produced a VHH-7D12-mouse-IgG-Fc fusion protein to detect the expression of EGFR in KYSE cell lines. This fusion protein was successfully utilized as a primary antibody for flow cytometry at varying concentrations. Meanwhile, we also generated an EGFR-mouse-IgG-Fc fusion protein to detected the expression of VHH-7D12 in our 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells. These findings support the potential of VHH-7D12 as an antigen recognition domain for CAR-T cells therapy. Interleukin-21 (IL-21), produced by various immune cells including CD4 + T cells and natural killer T cells, belongs to the common cytokine receptor γ chain family. IL-21 can promote the proliferation, survival, differentiation and function of T cells and enhance the anti-tumor ability of CD8 + T cells and natural kill cells[34]. IL-21 also can re-activate the exhausted cytotoxicity CD8 + T cells that residing in tumor microenvironment[35]. CAR-T cells that co-express hIL-21 perform better anti-tumor ability than CAR-T cells alone[36]. Additionally, IL-21 also can inhibit the development of regulatory T cells, which are known to impede tumor-infiltrating lymphocytes[37, 38]. In this study, human IL-21 cDNA was incorporated into the CAR construct following the CD3ζ sequence. The 7D12-CAR-hIL-21-T cells performed better proliferation ability than the 7D12-CAR-T cells and UTD groups. Meanwhile, more CD8 + T cells were detected in 7D12-CAR-hIL-21-T cells group. We also investigated more CD3+/CD8+/CD45RO + CD62L + central memory T cells in7D12-CAR-hIL-21-T cells that have been reported can prolong the survival and decreased the risk of recurrence in triple-negative breast cancer[39]. Although the proportion of CD4 + T cells has decreased, we detected that the percentage of CD3+/CD4+/CD45RO + CD62L + central memory T cells was increased in 7D12-CAR-hIL-21-T cells group. We suspected that this CD3+/CD4+/CD45RO + CD62L + T cells may include the increase of T helper (Th) cells, which may be a direction for subsequent research on the effect of IL-21 on CAR-T cell function. Furthermore, the modified CAR-T cells exhibited enhanced IFN-g secretion and improved anti-tumor ability when co-cultured with KYSE-30, KYSE-150 and KYSE-510 tumor cells. In vivo experiments confirmed the in vitro findings, showing effective tumor suppression and prolonged survival in KYSE-150 tumor-bearing mice treated with our CAR-T cells. To further evaluate the efficacy of the treatment, we assessed the secretion of IFN-g in the peripheral blood of 7D12-CAR-hIL-21-T cells treated mice at three different time points. While there was no significant difference in IFN-g secretion over time, the 7D12-CAR-hIL-21-T cells treated mice exhibited improved tumor suppression. Although EGFR is expressed at low levels in normal tissues, we observed no significant tissue damage in our mouse model, supporting the safety and potential of 7D12-hIL-21-CAR-T cells as a viable immunotherapy for ESCC. Materials and Methods Cell lines The human esophageal cancer cell lines KYSE-30, KYSE-70, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, and KYSE-510, along with the normal esophageal epithelial cell line HET-1A, were cultured in RPMI-1640 medium (GIBCO, Thermo Fisher Scientific, USA). Human T cells were maintained in X-VIVO 15 media (Lonza) supplemented with 100 IU/mL human IL-2 (PeproTech). All culture media were enriched with 10% (v/v) fetal bovine serum (FBS, Biological Industries, Israel) and 1% penicillin-streptomycin to support optimal growth conditions. Isolation and expansion of T cells Human peripheral blood mononuclear cells (PBMCs) were obtained from three healthy donors and isolated by density gradient centrifugation as previously described[40]. Human T cells were then isolated from PBMC by anti-human CD3/CD28 Dynabeads (Gibco) and cultured in X-VIVO 15 (LONZA) supplemented with 5%(v/v) fetal bovine serum, 100IU/ml human IL2 (Peprotech), 100 mM β-mercaptoethanol (Sigma) and 1% penicillin-streptomycin. CAR construction The lentiviral vectors encoding the 7D12-CAR and the 7D12-CAR-IL-21 constructs represent the second-generation CAR architecture. This design incorporates the human CD8 hinge and transmembrane domains, coupled with the intracellular co-stimulation domain of human 4-1BB and the CD3ζ signaling domain. The specificity for human EGFR was conferred by the anti-human-EGFR-VHH 7D12, as previously characterized by Rob C. Rovers et al[41]. A human CD8 signal peptide was engineered upstream of the 7D12 sequence. Untransduced (UTD) T cells were employed as a negative control within the experimental framework. Lentivirus production and T cells transduction All lentivirus of CAR were produced by 293T cells. Simply, the 293T cells were seeded at 6x10 6 cells per 10-cm dish 12 hours before transfection. The transfer plasmids that contain CARs sequence, the two package plasmids pMDLg/pRRE and pRSV-Rev, and the envelope plasmid pCMV-VSV-G were transfected simultaneously into 293T cells using polyethyleneimine (PEI) transfection in a ratio of 15:7.5:6:6. The medium was replaced 10 hours later and harvested at 48 and 72 hours after transfection. Harvested lentivirus particles were first removed impurities by filtration with a 0.45µm membrane and then concentrated by ultracentrifugation at 10,000 x g for 16 hours. All lentiviruses are resuspended with X-VIVO culture medium and frozen at -80°C. Human CD3 T cells were isolated and stimulated by anti-human CD3/CD28 beads at day 0. After 48 hours of invitro stimulation, 1x10 5 T cells were seeded with lentivirus at a multiplicity of infection (MOI) 5 in the presence of 100µg/ml Synperonic® F 108(Sigma) as assisted transfection reagents in a 24-well plate and infected at 32℃,1200xg for 90 minutes. The transduction medium was replaced another 4 hours later and then T cells were cultured with fresh medium every two days at cell density in 3-5x10 5 per ml. Flow cytometry assays For cell membrane stain, 5x10 5 cells were incubated with 2.5µg/ml fluorescent antibodies at 4℃ for 30 min protected from light. After culture, cells were washed with 1x wash buffer (BD bioscience) and then suspended for staining the second antibody as described before. The following antibodies were used in this topic: EGFR Monoclonal Antibody(H11) (thermos-fisher MA5-13070); Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H + L) (Beyotime A0428); MonoRab™ Rabbit Anti-Camelid VHH Cocktail [iFluor 488] (Genscript). The human T cells surface markers were detected using a FITC-conjugated mouse anti-humanCD3 antibody, Alexa Fluor 700-conjugated mouse anti-human CD4 antibody, Percp/cy5.5-conjugated mouse anti-human CD8 antibody, Brilliant Violet 785-conjugated mouse anti-human CD45RA antibody, Brilliant Violet 650-conjugated mouse anti-human CD45RA antibody, PE-conjugated mouse anti-human CD197 (CCR7) antibody, Brilliant Violet 421-conjugated mouse anti-human CD45RO antibody and PE-CY7-conjugated mouse anti-human CD27 antibody (all from BioLegend). Cytotoxicity assays The Cytotoxicity LDH Assay Kit-WST (DOJINDO CK12) was used to detect the LDH secretion of KYSE cell lines that co-cultured with CAR-T cells or UTD T cells at different ratios (T cells: target cells = 2:1, 4:1, 8:1, 16:1, 32:1). After 16 hours, the culture supernatants were collected and detected by using LDH release assay, human IFN-γ ELISA kit (MultiSciences 70-EK1804) and human TFN-α ELISA kit (MultiSciences 70-EK182) respectively. Measurement of human IL-21 The human IL-21 Uncoated ELISA Kit with plates (Invitrogen 88-8218-86) was used to measure IL-21 concentrations. Briefly, 1x106 CAR-T cells and UTD T cells were cultured in 1mL X-VIVO medium with 5%(v/v) fetal bovine serum. Cell culture supernatants were collected and assayed at 24 hours. The detected of hIL-21 was performed according to the manufacturer’s instructions of the human IL-21 Uncoated ELISA Kit. Bulk RNA-sequencing RNA-seq was performed by Shanghai OE Biotech Co., Ltd. Briefly, total RNA was extracted from T cells that cultured invitro including 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells using mirVana™ miRNA ISOlation Kit, (Ambion-1561). Briefly, RNA integrity was assessed by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina, RS-122-2101), total 4 µg RNA was used to build library construction. Next, the libraries underwent sequencing via the Illumina HiSeq X Ten sequencing platform. These 150bp paired-end reads generated as raw data. The initial data underwent purification to obtain high-quality, clean data. Thereafter, the clean data was utilized to acquire in-depth transcriptional information following the human genome (NCBI_GRCh38.p13). GO and KEGG pathway analyses were conducted on genes meeting the criteria of ± 2-fold change and corrected P-value < 0.05. Xenograft tumor models For the establishment of KYSE-150 xenograft tumor models, 6-week-old NOD/ SCID mice were inoculated s.c. with 5x10 6 KYSE-150 cells on the right flank. When the tumor size was ~ 100 mm 3 in approximately 7 days, the mice were divided randomly into 5 groups (6 in each group), injected with 5x10 6 T cells dissolved in 100µl control PBS,100µl PBS including UTD T cells, 7D12-CAR-T cells or 7D12-CAR-hIL-21-T cells respectively. All of these cells were injected through the tail vein (i.v.) on day 1. Tumor size was detected every two days until the tumor size reached 100 mm3 when these mice were sacrificed. All animals cultured and treated were in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals and carried out in Laboratory Animal Center, Zhengzhou University. Statistical Analysis Statistical analysis was performed using Graph Pad Prism 6 and IBM SPSS statistical software (version 19.0). Student’s t-test was utilized to compare differences between two groups, while a one-way ANOVA test was used to analyze differences among multiple groups. Additionally, Kaplan–Meier survival analysis was employed to assess differences in survival. Statistical significance was considered when the p-values were at or below 0.05. Data availability The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Declarations Competing interests The authors declare no competing interests. Author Contribution C.L.Z, L.H. and Z.M.W. conceptualized the study. C.L.Z, Y.Y.L, H.R.G and D.Y.Z. performed experiments. C.L.Z, Y.P and X.Q.L. analyzed the data and generated the figures. S.S.L, L.H. and Z.M.W. interpreted data and reviewed the manuscript. C.L.Z, L.H. and Z.M.W. wrote the manuscript with input from all authors. Acknowledgements This study was funded by research funding from the General Program of Henan Natural Science (222300420531). Data Availability Data is provided within the manuscript or supplementary information files. References Abnet CC, Arnold M, Wei WQ: Epidemiology of Esophageal Squamous Cell Carcinoma . Gastroenterology 2018, 154 (2):360–373. Zhu H, Ma X, Ye T, Wang H, Wang Z, Liu Q, Zhao K: Esophageal cancer in China: Practice and research in the new era . Int J Cancer 2023, 152 (9):1741–1751. 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Sabatino M, Hu J, Sommariva M, Gautam S, Fellowes V, Hocker JD, Dougherty S, Qin H, Klebanoff CA, Fry TJ et al : Generation of clinical-grade CD19-specific CAR-modified CD8 + memory stem cells for the treatment of human B-cell malignancies . Blood 2016, 128 (4):519–528. Martinez M, Moon EK: CAR T Cells for Solid Tumors: New Strategies for Finding, Infiltrating, and Surviving in the Tumor Microenvironment . Front Immunol 2019, 10 :128. Sabbah DA, Hajjo R, Sweidan K: Review on Epidermal Growth Factor Receptor (EGFR) Structure, Signaling Pathways, Interactions, and Recent Updates of EGFR Inhibitors . Curr Top Med Chem 2020, 20 (10):815–834. Sigismund S, Avanzato D, Lanzetti L: Emerging functions of the EGFR in cancer . Mol Oncol 2018, 12 (1):3–20. Voldborg BR, Damstrup L, Spang-Thomsen M, Poulsen HS: Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials . Ann Oncol 1997, 8 (12):1197–1206. Sun S, Ding Z, Yang X, Zhao X, Zhao M, Gao L, Chen Q, Xie S, Liu A, Yin S et al : Nanobody: A Small Antibody with Big Implications for Tumor Therapeutic Strategy . Int J Nanomedicine 2021, 16 :2337–2356. Condeminas M, Macias MJ: Overcoming challenges in structural biology with integrative approaches and nanobody-derived technologies . Curr Opin Struct Biol 2024, 84 :102764. Safarzadeh Kozani P, Naseri A, Mirarefin SMJ, Salem F, Nikbakht M, Evazi Bakhshi S, Safarzadeh Kozani P: Nanobody-based CAR-T cells for cancer immunotherapy . Biomark Res 2022, 10 (1):24. Rossotti MA, Trempe F, van Faassen H, Hussack G, Arbabi-Ghahroudi M: Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries . Methods Mol Biol 2023, 2702 :107–147. de Bruijn HS, Mashayekhi V, Schreurs TJL, van Driel P, Strijkers GJ, van Diest PJ, Lowik C, Seynhaeve ALB, Hagen T, Prompers JJ et al : Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging . Theranostics 2020, 10 (5):2436–2452. Chabab G, Bonnefoy N, Lafont V: IL-21 Signaling in the Tumor Microenvironment . Adv Exp Med Biol 2020, 1240 :73–82. Batra SA, Rathi P, Guo L, Courtney AN, Fleurence J, Balzeau J, Shaik RS, Nguyen TP, Wu MF, Bulsara S et al : Glypican-3-Specific CAR T Cells Coexpressing IL15 and IL21 Have Superior Expansion and Antitumor Activity against Hepatocellular Carcinoma . Cancer Immunol Res 2020, 8 (3):309–320. Chang L, Wu H, Huang W, Li Y, Chen Y, Li X, Yao Z, Chen X, Lai X, Zheng R et al : IL-21 induces pyroptosis of Treg cells via Akt-mTOR-NLRP3-caspase 1 axis in eosinophilic chronic rhinosinusitis . J Allergy Clin Immunol 2023, 152 (3):641–655.e614. Tanaka A, Sakaguchi S: Regulatory T cells in cancer immunotherapy . Cell Res 2017, 27 (1):109–118. Sun X, Zhai J, Sun B, Parra ER, Jiang M, Ma W, Wang J, Kang AM, Kannan K, Pandurengan R et al : Effector memory cytotoxic CD3(+)/CD8(+)/CD45RO(+) T cells are predictive of good survival and a lower risk of recurrence in triple-negative breast cancer . Mod Pathol 2022, 35 (5):601–608. Yi PC, Zhuo L, Lin J, Chang C, Goddard A, Yoon OK: Impact of delayed PBMC processing on functional and genomic assays . J Immunol Methods 2023, 519 :113514. Roovers RC, Vosjan MJ, Laeremans T, el Khoulati R, de Bruin RC, Ferguson KM, Verkleij AJ, van Dongen GA, van Bergen en Henegouwen PM: A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth . Int J Cancer 2011, 129 (8):2013–2024. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pdf Cite Share Download PDF Status: Posted Version 1 posted 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-4485969","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":317727571,"identity":"f029e6da-3174-4753-829a-e6810a6ac878","order_by":0,"name":"Chenglin Zhang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chenglin","middleName":"","lastName":"Zhang","suffix":""},{"id":317727572,"identity":"8f3e76fb-fcaa-442e-b9e1-12fc3a7b8b35","order_by":1,"name":"Yanyan Liu","email":"","orcid":"","institution":"Affiliated Cancer Hospital of Zhengzhou University \u0026 Henan Cancer 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15:17:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4485969/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4485969/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59019036,"identity":"553e934f-2856-466d-a728-aa969d799a39","added_by":"auto","created_at":"2024-06-25 11:23:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":334962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of EGFR Expression in Esophageal Squamous Cell Carcinoma (ESCC) Cell Lines \u003c/strong\u003e(A) Flow cytometry analysis of EGFR expression across nine ESCC cell lines (KYSE-30, KYSE-70, KYSE-140, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, and KYSE-510) compared to human normal esophageal epithelial cells (HET-1A), illustrating differential expression levels of EGFR.\u003cstrong\u003e \u003c/strong\u003e(B) Mean fluorescence intensity (MFI) of EGFR expression was normalized to facilitate comparison across cell lines.\u003cstrong\u003e \u003c/strong\u003e(C) Bar chart representation of the normalized MFI, providing a visual comparison of EGFR expression levels among the ESCC cell lines and HET-1A cells.\u003cstrong\u003e \u003c/strong\u003e(D) mRNA expression levels of EGFR in the nine ESCC cell lines, highlighting the variability in EGFR transcription across different cell lines.\u003c/p\u003e","description":"","filename":"Onlinefigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/4b6cee15042f159d6f977a72.png"},{"id":59018636,"identity":"ff2b2428-ceec-4b61-8fdb-42347d341b5b","added_by":"auto","created_at":"2024-06-25 11:15:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393193,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic representations of the constructs used for CAR-T cell generation. Top: Expression vector for the EGFR Met1-Ser645-mIgG-Fc fusion protein. Middle: Lentiviral vector encoding the EGFR-VHH-7D12-CAR. Bottom: Lentiviral vector encoding the EGFR-VHH-7D12-P2A-hIL-21-CAR, illustrating the gene architecture. (B) Western blot analysis demonstrating the presence of EGFR \u003csub\u003eMet1-Ser645\u003c/sub\u003e-mIgG-Fc protein in DMEM medium, the purified EGFR \u003csub\u003eMet1-Ser645\u003c/sub\u003e-mIgG-Fc protein, and a 10-fold concentration of the EGFR Met1-Ser645-mIgG-Fc protein, showcasing the protein's expression and purification efficiency. (C) Flow cytometry analysis of EGFR-VHH-7D12 expression in UTD T cells, 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells at a multiplicity of infection (MOI) of 5. (D) Flow cytometry analysis of EGFR-VHH-7D12 expression in 7D12-CAR-T cells that dual-stained with EGFR \u003csub\u003eMet1-Ser645\u003c/sub\u003e-mIgG-Fc and anti-mouse-IgG-Fc-FITC or stained with commercial anti-VHH cocktail-FITC antibody\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment and Characterization of EGFR-Specific Nanobody CAR-T Cells\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/18f7805faf36bc585cc90827.png"},{"id":59018635,"identity":"63123c6e-5e7b-4eef-8182-e02451c3dc57","added_by":"auto","created_at":"2024-06-25 11:15:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":901418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe introduction of human IL-21 can influence the subgroup of CAR-T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) ELISA quantification of human IL-21 production in vitro by UTD T cells, 7D12-CAR-T cells, and 7D12-CAR-hIL-21-T cells from three healthy donors, indicating the secretion levels of IL-21 following CAR transduction. (B) Growth curves of UTD T cells, 7D12-CAR T cells, and 7D12-CAR-hIL-21 T cells over an 11-day in vitro culture period. (C) Flow cytometry detected the CD4 and CD8 in EGFR-VHH-7D12-CAR and EGFR-VHH-7D12-P2A-hIL-21-CAR in vitro cultured 7 days. (D) The histogram shows the change in the proportion of CD4. (E) The histogram shows the change in the proportion of CD8. (F) Flow cytometry detected the CD45RO and CD62L in CD4 and CD8 T cells of EGFR VHH-7D12-CAR and EGFR-VHH-7D12-P2A-hIL-21-CAR T cells (G) The histogram shows the CD45RO+ CD62L+ T cells in the proportion of CD4+T cells. (H) The histogram shows the CD45RO+ CD62L+ T cells in the proportion of CD8+T cells. Values are presented as mean ± SEM. *, P \u0026lt; 0.05; **, P \u0026lt; 0.01，***, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/ba53d038834957f69df7ae4b.png"},{"id":59018638,"identity":"8283035e-c291-433e-b740-fef68661ad26","added_by":"auto","created_at":"2024-06-25 11:15:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":624945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn Vitro Cytotoxicity of EGFR-CAR T Cells Against ESCC Cell Lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Lactate dehydrogenase (LDH) release assay demonstrating the cytotoxicity of UTD T cells, EGFR-VHH-7D12-CAR T cells, and EGFR-VHH-7D12-P2A-hIL-21-CAR T cells when cocultured with ESCC cell lines KYSE-30, KYSE-150, and KYSE-510. The cells were incubated at various effector-to-target (E/T) ratios (32:1, 16:1, 8:1, 4:1, 2:1, and 1:1) at 37°C for 16 hours. The assay quantitatively measures the cytotoxic effect of CAR T cells on target ESCC cells through the detection of LDH released from lysed cells. (D-F) Interferon-gamma (IFN-γ) ELISA assay results following a 16-hour coculture of CAR T cells with KYSE-30, KYSE-150, and KYSE-510 cells at an E/T ratio of 16:1 at 37°C. The assay measures the secretion of IFN-γ by CAR T cells, indicating an immune response against the target ESCC cells. (G-I) TNF-α ELISA assay results after a 16-hour coculture of CAR T cells with KYSE-30, KYSE-150, and KYSE-510 at an E/T ratio of 16:1 at 37°C. The data are presented as mean ± SEM, with statistical significance indicated as *, P \u0026lt; 0.05; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/1d28b43d7640d2c746c818ad.png"},{"id":59019037,"identity":"ed3782eb-db4c-44d2-b066-8945c480efbd","added_by":"auto","created_at":"2024-06-25 11:23:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":510856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic Efficacy of EGFR-CAR T Cells in ESCC Cell Mouse Xenograft Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the experimental setup for evaluating the efficacy of EGFR-CAR T cell therapy in vivo. NSG mice were subcutaneously injected with KYSE-150 cells. Upon reaching a tumor volume of approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, mice were intravenously administered PBS as a negative control, 5x10\u003csup\u003e6\u003c/sup\u003e UTD T cells, 5x10\u003csup\u003e6\u003c/sup\u003e 7D12-CAR-T cells, or 5x10\u003csup\u003e6\u003c/sup\u003e 7D12-hIL-21-CAR-T cells. (B) Graph showing the progression of tumor volume measured twice weekly. This data illustrates the comparative tumor suppression capabilities of each treatment group over time. (C) Kaplan-Meier (KM) survival analysis of the overall survival rates of mice in each treatment group, providing insight into the potential life-prolonging effects of CAR-T cell therapies. (D) IFN-γ ELISA assay results detailing the concentration of IFN-γ in the serum of mice at 3-, 7-, and 15-days post-CAR-T cell injection. (E) Human IL-21 (hIL-21) ELISA assay results showing the concentration of hIL-21 in the serum of mice at 3-, 7-, and 15-days post-CAR-T cell injection. The presence of hIL-21 serves as an indicator of the activity of 7D12-hIL-21-CAR-T cells. Data are presented as mean ± SEM, with statistical significance denoted as * P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001; **** P \u0026lt; 0.0001. volume reduction, improved survival rates, and increased cytokine production in treated mice.\u003c/p\u003e","description":"","filename":"Onlinefigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/a8555e21ac6515d392a76ca3.png"},{"id":59018640,"identity":"04c75c8d-32c2-435d-98e4-97468a5de242","added_by":"auto","created_at":"2024-06-25 11:15:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3361696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessing the Safety of EGFR-CAR T Cells in ESCC Cell Mouse Xenograft Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Hematoxylin and eosin (HE) staining of major organ tissues (heart, liver, spleen, lung, and kidney) from mice treated with 7D12-CAR-T cells and 7D12-hIL21-CAR-T cells. The images demonstrate the absence of histopathological damage, indicating that the CAR-T cell treatments did not induce tissue injury in these organs. (B) PCR analysis for the detection of residual genomic DNA from CAR-T cells in the heart, liver, spleen, lung, and kidney tissues. This assay was utilized to investigate the potential infiltration and persistence of CAR-T cells in non-target organs. (C-E) Serum levels of liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured to evaluate liver function and the potential hepatotoxicity associated with CAR-T cell therapy. The results, denoted as \"NS\" means no significance.\u003c/p\u003e","description":"","filename":"Onlinefigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/fbe9df9c33b586cc3086df23.png"},{"id":61209614,"identity":"bc1e2308-92d1-4961-9506-7766cd72aca4","added_by":"auto","created_at":"2024-07-27 06:40:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3011036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/355d2c2a-caf6-4ebc-b588-3424c917a2bb.pdf"},{"id":59018634,"identity":"15d3f6fd-7f30-4ea7-bd94-0203fa6d0052","added_by":"auto","created_at":"2024-06-25 11:15:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1481380,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485969/v1/16ab3597837385be7b1b9743.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a new EGFR-VHH-CAR T-cell therapy for treatment of esophageal squamous cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer among the top 10 most prevalent malignancies globally, characterized by high morbidity and mortality rates [1]. Esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) are two primary subtypes of esophageal cancer. Notably, China harbors over half of the global esophageal cancer patient population, with ESCC constituting more than 90% of these cases[2]. ESCC is particularly aggressive, exhibiting rapid progression, a high recurrence rate, and a dismal 5-year survival rates below 30%, underscoring the urgency for advanced clinical research[3]. Conventional systemic chemotherapy, typically involving cisplatin and 5-fluorouracil (5-FU) (CF), remains the standard treatment for ESCC [4, 5]. However, the limited anti-tumor efficacy and poor prognosis have necessitated the exploration of more effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eEGFR, a receptor tyrosine kinase abundantly expressed on ESCC cells and other epithelial-derived tumors with poor prognosis, serves as a target for CAR-T cell therapy[6]. Its overexpression in ESCC patients, contrasted with low expression in normal tissues, positions EGFR as a prime therapeutic target[7]. Previous clinical trials involving EGFR-targeted CAR-T cell therapy in various cancers, including triple-negative breast cancer, head and neck cancer, glioblastoma, and non-small cell lung cancer, have shown promising outcomes[8\u0026ndash;11].\u003c/p\u003e \u003cp\u003eChimeric antigen receptor-modified T (CAR-T) cells are engineered by introducing a single-chain fragment variable (ScFv) and an intracellular co-stimulatory domain, such as 4-1BB and CD3ζ to activate and enhance the cytotoxicity of T cells[12]. These modified T cells can specifically target and eliminate tumor cells without the need for major histocompatibility complex (MHC) presentation[13]. However, challenges with ScFv, including improper folding and aggregation, have limited the efficacy of CAR-T cells, prompting the search for alternatives[14].\u003c/p\u003e \u003cp\u003eThe variable domain of heavy chain of heavy-chain antibodies (VHHs), also named nanobodies, emerges as a promising substitute for ScFv due to their small size (15kDa), which facilitates binding to complex, and their reduced immunogenicity thanks to humanization modification[15]. VHH-based CAR-T therapy, particularly targeting B cell maturation antigen (BCMA), has been FDA-approved for its potent tumor-suppressive function in multiple myeloma (MM), achieving an 88% overall response (OR) and 68% complete response (CR)[16].\u003c/p\u003e \u003cp\u003eIn this research, we introduced the EGFR-VHH-7D12, a nanobody targeting the third domain of EGFR with high binding affinity, offering potential application in the for EGFR-targeted VHH-based CAR-T therapy[17]. Furthermore, we investigated human interleukin 21, a multifunctional cytokine from the common g chain family, known for its role in promoting CD8\u0026thinsp;+\u0026thinsp;killer T cell proliferation and enhancing interferon-gamma (IFN‐γ) secretion[18, 19]. Unlike lIL-2, lIL-7, and lIL-15, IL-21 activates signal transducer and activators of transcription 1(STAT1) and 3 (STAT3), but not STAT5A or STAT5B, indicating its unique influence on T cell anti-tumor functions[20].\u003c/p\u003e \u003cp\u003eHere, we developed two novel nanobody-based CAR-T cells: 7D12-CAR-T cells (7D12-CAR) and 7D12-CAR-T cells expressing human IL-21(7D12-CAR-hIL-21). Both constructs specifically target EGFR-positive ESCC cell lines, with the latter demonstrating enhanced persistence in expressing human IL-21 and superior anti-tumor efficacy in vitro and in vivo.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEGFR was Upregulated in ESCC with Predicted Poor Prognosis\u003c/h2\u003e \u003cp\u003eThe aberrantly activated tyrosine kinase pathway, closely linked to EGFR, plays a pivotal role in promoting anti-apoptosis and carcinogenesis in normal epithelial cells. Notably, EGFR's aberrant expression is a common feature in epithelial tissue malignancies, correlating strongly with poor prognosis, particularly in esophageal squamous cell carcinoma (ESCC)[21]. Given the variable EGFR expression across different cell lines, we embarked on a comparative analysis of EGFR levels in various KYSE cell lines (KYSE-30, KYSE-70, KYSE-140, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, KYSE-510) and normal human esophageal epithelial cells (HET-1A) using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur findings revealed a prevalent overexpression of EGFR in KYSE cell lines, albeit with varying intensities, in contrast to its lower expression in normal human esophageal epithelial cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Subsequent mRNA expression analysis of EGFR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) corroborated the flow cytometry outcomes, particularly for KYSE-30, KYSE-150, and KYSE-510, which demonstrated high, medium, and low levels of EGFR expression, respectively. for further investigate the efficacy of our CAR-T cells, we selected these three KYSE cell lines (KYSE-30, KYSE-150, and KYSE-510) based on their distinct EGFR expression levels for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of EGFR- specific VHH-7D12 nanobody CAR-T cells\u003c/h2\u003e \u003cp\u003eThe nanobody EGFR-VHH-7D12, recognized for its ability to specifically target the membrane surface protein EGFR, has been increasingly utilized in immunotherapy applications over the past few years[17]. IL-21, a potent immunoregulatory cytokine, is instrumental in the immune response, enhancing the activation and proliferation of T cells and natural killer cells [22, 23]. To assess the specificity of EGFR-VHH-7D12, we designed an extracellular segment of EGFR protein EGFR \u003csub\u003eMet1\u0026minus;Ser645\u003c/sub\u003e-mouse IgG Fc fusion protein (eEGFR-mIgG-Fc). The architecture of this fusion protein is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA (top panel). The EGFR-VHH-7D12 was employed as an alternative to ScFv to construct the 7D12-CAR, incorporating EGFR-VHH-7D12, CD8 hinge and transmembrane domains, 4-1BB, and CD3zeta domains. Subsequently, the 7D12-CAR-hIL-21 variant was engineered by appending a 2A peptide and human IL-21 subsequent to the CD3ζ intracellular signaling domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, middle and bottom panels).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the production of the EGFR \u003csub\u003eMet1\u0026minus;Ser645\u003c/sub\u003e-mIgG-Fc protein, 293E cells were utilized, followed by concentration of the detection protein using a 50KD ultrafiltration tube at 1500 xg. The medium, EGFR \u003csub\u003eMet1\u0026minus;Ser645\u003c/sub\u003e-mIgG-Fc infusion protein, and a 10-fold concentrated version of the EGFR \u003csub\u003eMet1\u0026minus;Ser645\u003c/sub\u003e-mIgG-Fc infusion protein were subsequently analyzed via Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Supplemental Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTo assess the expression of EGFR-VHH-7D12, we utilized the commercial anti-VHH cocktail-FITC antibody, targeting the conserved sequence of VHH, to investigated the stability of EGFR-VHH-7D12 expression across T cells from three different donors. Flow cytometry analysis revealed that T cells, at a multiplicity of infection (MOI) of 5, exhibited EGFR-VHH-7D12 expression on the surface of both 7D12-CAR-T and 7D12-CAR-hIL-21-T cells, but not on UTD T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Further, we investigated the recognition ability of EGFR-VHH-7D12 by utilizing the concentrated EGFR \u003csub\u003eMet1\u0026minus;Ser645\u003c/sub\u003e-mIgG-Fc infusion protein as a primary antibody in both untransduced (UTD) T cells and 7D12-CAR-T cells. For comparison, commercial anti-VHH cocktail-FITC antibody served as the positive control. The combination of EGFR \u003csub\u003eMet1\u0026minus;Ser645\u003c/sub\u003e-mIgG-Fc infusion protein with anti-mouse IgG-Fc-FITC enabled the detection of EGFR-VHH-7D12 expression in 7D12-CAR-T cells. Notably, the recognition rate was slightly better compared to the commercial antibody (86.3% vs. 84.4%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Neither antibody yielded positive results in UTD T cells (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating the specificity of EGFR-VHH-7D12 for EGFR protein and validating our EGFR \u003csub\u003eMet1\u0026minus;Ser645\u003c/sub\u003e-mIgG-Fc infusion protein's ability to specifically target EGFR on the cell surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings collectively demonstrate the specific targeting capability of EGFR-VHH-7D12 towards EGFR protein and the successful generation of EGFR-specific VHH-7D12 nanobody CAR-T cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of Human IL-21 on CAR-T Cell Subpopulations\u003c/h2\u003e \u003cp\u003eIL-21 has been documented to significantly affect various T cell cultures. To determine the impact of IL-21 in CAR-T cells proliferation and differentiation, we first detected the expression of IL-21 in 7D12-CAR-hIL-21-T cells. The secretion of hIL-21 by 7D12-CAR-hIL-21-T cells from all three donors was quantified, exceeding 2000 pg/ml at an MOI of 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The influence of hIL-21 on promoting CAR-T cell proliferation in vitro was subsequently examined. Results indicated that 7D12-CAR-hIL-21-T cells exhibited enhanced proliferative capabilities compared to both UTD T cells and 7D12-CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eOur study further elucidates its impact on CAR-T cell subpopulations. After 11 days of in vitro culture, 7D12-CAR-hIL-21-T cells exhibited a decrease in CD4\u0026thinsp;+\u0026thinsp;T cell proportion (50.27%\u0026plusmn;0.484% vs. 63.40%\u0026plusmn;0.866%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and an increase in CD8\u0026thinsp;+\u0026thinsp;T cells (40.73%\u0026plusmn;0.491% vs. 28.07%\u0026plusmn;0.066%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to 7D12-CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E). This shift suggests IL-21's role in modulating the CD4+/CD8\u0026thinsp;+\u0026thinsp;T cell ratio within the CAR-T cell milieu.\u003c/p\u003e \u003cp\u003eMoreover, we investigated the memory T cell subsets between 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells. The latter group demonstrated a higher expression of CD3+/CD4+/CD45RO\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;central memory T cells (83.67%\u0026plusmn;1.898% vs. 67.8% \u0026plusmn; 0.2517%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and CD3+/CD8+/CD45RO\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;central memory T cells (55.63%\u0026plusmn;0.6984% vs. 43.93%\u0026plusmn;0.4910%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to 7D12-CAR-T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003eTo delve deeper into hIL-21's functional impact on CAR-T cells, we performed transcriptome analysis on both 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells after 11 days of in vitro culture. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified the MAPK signaling pathway as significantly modulated, which may enhance the proliferation of CAR-T cells (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA)[24]. Further analysis of genes related to the MAPK signaling pathway, with a log2 scale cutoff of 0.58, was visualized through a heatmap, delineating the gene expression changes within this pathway (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese comprehensive analyses suggest that IL-21 plays a pivotal role in enhancing the proliferation ability and skewing the CAR-T cell population towards a more cytotoxic phenotype, with increased proportions of CD8\u0026thinsp;+\u0026thinsp;T cells and CD3+/CD8+/CD45RO\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;central memory cytotoxic T cells. Additionally, an increase in CD3+/CD4+/CD45RO\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;central memory T cells was observed and the change of MAPK signaling pathway, highlighting IL-21's broad immunomodulatory effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro Cytotoxicity of EGFR-CAR T Cells Against ESCC Cell Lines\u003c/h2\u003e \u003cp\u003eEGFR is prevalently overexpressed in esophageal squamous cell carcinomas (ESCCs), highlighting its potential as a therapeutic target[23]. To evaluate the antitumor efficacy of 7D12-CAR-T and 7D12-CAR-hIL-21-T cells against ESCC with varying EGFR expression levels, we utilized KYSE-30, KYSE-150, KYSE-510, and an EGFR-knockout cell line as targets, as detailed by western blot (Supplemental Fig.\u0026nbsp;5A). Both CAR-T cell variants demonstrated specific and potent cytotoxicity against the KYSE-30, KYSE-150, and KYSE-510 cell lines (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). In contrast, cytotoxic activity was negligible against the EGFR-knockout cell line, indicating the specificity of the CAR-T cells' action (Supplemental Fig.\u0026nbsp;5B). Remarkably, 7D12-CAR-hIL-21-T cells outperformed their counterparts in lysing all three ESCC cell lines, underlining the enhanced efficacy conferred by hIL-21. Furthermore, we assessed the secretion of cytotoxic cytokines, IFN-γ and TNF-α, during co-culture with tumor cells at an effector-to-target (E:T) ratio of 16:1. 7D12-CAR-hIL-21-T cells exhibited elevated levels of IFN-γ secretion compared to 7D12-CAR-T cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F), while TNF-α levels remained comparable across both CAR-T cell types (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003eThese findings underscore the nanobody-based CAR-T cells' robust and consistent antitumor efficacy across different ESCC cell lines. Additionally, the incorporation of hIL-21 appears to augment the anti-tumor potency of CAR-T cells, potentially through the upregulation of IFN-γ expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of EGFR-CAR T Cells in ESCC Cell Mouse Xenograft Models\u003c/h2\u003e \u003cp\u003eThis study evaluated the therapeutic potential of 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells against esophageal squamous cell carcinoma (ESCC) growth in mouse xenograft models. Non-obese diabetic/severe combined immunodeficient/γ-chain\u0026minus;/\u0026minus; (NSG) mice were subcutaneously inoculated with 5.0x10^6 KYSE-150 cells. Upon tumor volumes reaching approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, mice were administered 5.0x10\u003csup\u003e6\u003c/sup\u003e CAR-T cells, 5.0x10\u003csup\u003e6\u003c/sup\u003e untransduced (UTD) T cells, or phosphate-buffered saline (PBS) in a 100\u0026micro;l volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Our observations revealed that both CAR-T cell variants significantly inhibited tumor proliferation and enhanced survival rates, outperforming the UTD T cells and PBS control groups. Among them, the 7D12-CAR-hIL-21-T cells displayed markedly superior tumor growth suppression compared to both the UTD T cells and 7D12-CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A minor reduction in body weight was noted in the initial phase of 7D12-CAR-T cell treatment; however, no significant weight loss was observed throughout the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Cytokine analysis in peripheral blood showcased sustained hIL-21 expression exclusively in the 7D12-CAR-hIL-21-T cell treatment group, with no appreciable difference in IFN-γ levels between the two CAR-T cell groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, the presence of hIL-21 was confirmed in the peripheral blood of the 7D12-CAR-hIL-21-T cell group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results underscore the effectiveness of EGFR-VHH CAR-T cell therapies in curtailing the growth of KYSE-150 subcutaneous tumors, with the 7D12-CAR-hIL-21-T cells demonstrating enhanced therapeutic efficacy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSafety Profile of EGFR-CAR T Cells in ESCC Cell Mouse Xenograft Models\u003c/h2\u003e \u003cp\u003eGiven that EGFR serves as a tumor-associated antigen, evaluating the on-target, off-tumor (OTOT) risk associated with EGFR-VHH-7D12 CAR-T cell therapy is imperative[23]. Our initial investigation focused on potential tissue damage in mouse xenograft models treated with phosphate-buffered saline (PBS), 7D12-CAR-T cells, and 7D12-CAR-hIL-21-T cells. Immunohistochemical analysis across these groups revealed no significant tissue damage in critical organs, including the heart, liver, spleen, lung, and kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite the minimal EGFR expression in these tissues, we further examined the potential infiltration of CAR-T cells into normal tissues. Genomic DNA extracted from major organs was subjected to PCR amplification using WPRE-specific primers. WPRE sequences were detected in the 7D12-CAR-T and 7D12-CAR-hIL-21-T cell treatment groups, but not in the PBS control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). While immunohistochemistry did not indicate overt tissue damage, the presence of CAR-T cells in various tissues suggests a potential OTOT risk. Additionally, we assessed liver function by measuring levels of liver enzymes (ALT, AST, ALP) to identify any signs of liver damage. The results showed no significant differences in enzyme levels between the CAR-T cell therapy groups and the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E).\u003c/p\u003e \u003cp\u003eThese findings collectively suggest that EGFR-VHH-7D12-CAR-T cell therapy does not induce significant tissue damage, despite evidence of CAR-T cell presence in non-target tissues. This comprehensive safety assessment underscores the potential of VHH-7D12-based CAR-T cell therapy for clinical application, highlighting its favorable safety profile in the context of ESCC treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChimeric antigen receptor (CAR) -modified T cells have demonstrated remarkable efficacy in identifying and destroying tumor cells, particularly in hematological malignancies[25]. However, their anti-tumor capability in solid tumors, including ESCC, remains limited[26]. In our study, we developed a new nanobody-based anti-EGFR-CAR-T cells, incorporating hIL-21 to enhance their anti-tumor activity against ESCC. Our findings indicate that both 7D12-CAR-T cells, and 7D12-CAR-hIL-21-T cells can effectively inhibit tumor growth. Notably, the 7D12-CAR-hIL-21-T cells performed superior proliferation, increased memory T cells subgroups, and more potent anti-tumor effects both in vitro and in vivo. These results underscore the potential of our nanobody-based anti-EGFR-CAR-T cells, especially those expressing hIL-21, as a promise treatment for ESCC, offering a new immunotherapeutic strategy for this aggressive cancer.\u003c/p\u003e \u003cp\u003eThe Epidermal Growth Factor Receptor (EGFR), part of the epidermal growth factor receptor family, is commonly present on epithelial-derived tissue cell membranes [27, 28]. EGFR activation, through ligand binding and subsequent dimerization, initiates signaling cascades that promote cell proliferation, angiogenesis, invasion, metastasis, and apoptosis inhibition[29]. EGFR-targeted CAR-T cells have shown efficacy in various tumor types, including triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), multiple glioblastoma (GBM), and ESCC[8\u0026ndash;11]. High expression of EGFR is associated with poor prognosis[21]. In this study, we assessed EGFR expression across nine ESCC cell lines, selecting KYSE-30, KYSE-150, and KYSE-510 to represent varying EGFR expression levels. Both 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells successfully targeted and eliminated these three tumor cell lines in vitro.\u003c/p\u003e \u003cp\u003eIn 1993, researchers identified a naturally occurring heavy-chain antibody in dromedary camels, known as heavy-chain antibodies (HCAb), which lack light chains[30]. Through bioengineering, the heavy chain variable region domain of HCAb can be cloned to create VHH antibodies, also called nanobodies[31]. Compared to scFv, nanobody-based CAR-T cells exhibit superior characteristics, such as lower immunogenicity, easier folding outside the cell membrane, reduced likelihood of abnormal aggregation, and enhanced stability[32]. Carvykti, a BCMA-targeted nanobody-based CAR-T cell, is the first FDA-approved commercial nanobody CAR-T product, signaling the strong potential of nanobodies in CAR-T cell therapy[33]. In this study, novel nanobody-based CAR-T cells targeting EGFR were developed based on the VHH sequence 7D12 (EGFR-VHH-7D12). This VHH have been applied in photodynamic therapy (PDT) by conjugated with photosensitizing chemical substance (7D12-PS)[34]. This 7D12-PS performed high anti-tumor ability without damaging normal tissues. We designed and produced a VHH-7D12-mouse-IgG-Fc fusion protein to detect the expression of EGFR in KYSE cell lines. This fusion protein was successfully utilized as a primary antibody for flow cytometry at varying concentrations. Meanwhile, we also generated an EGFR-mouse-IgG-Fc fusion protein to detected the expression of VHH-7D12 in our 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells. These findings support the potential of VHH-7D12 as an antigen recognition domain for CAR-T cells therapy.\u003c/p\u003e \u003cp\u003eInterleukin-21 (IL-21), produced by various immune cells including CD4\u0026thinsp;+\u0026thinsp;T cells and natural killer T cells, belongs to the common cytokine receptor γ chain family. IL-21 can promote the proliferation, survival, differentiation and function of T cells and enhance the anti-tumor ability of CD8\u0026thinsp;+\u0026thinsp;T cells and natural kill cells[34]. IL-21 also can re-activate the exhausted cytotoxicity CD8\u0026thinsp;+\u0026thinsp;T cells that residing in tumor microenvironment[35]. CAR-T cells that co-express hIL-21 perform better anti-tumor ability than CAR-T cells alone[36]. Additionally, IL-21 also can inhibit the development of regulatory T cells, which are known to impede tumor-infiltrating lymphocytes[37, 38]. In this study, human IL-21 cDNA was incorporated into the CAR construct following the CD3ζ sequence. The 7D12-CAR-hIL-21-T cells performed better proliferation ability than the 7D12-CAR-T cells and UTD groups. Meanwhile, more CD8\u0026thinsp;+\u0026thinsp;T cells were detected in 7D12-CAR-hIL-21-T cells group. We also investigated more CD3+/CD8+/CD45RO\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;central memory T cells in7D12-CAR-hIL-21-T cells that have been reported can prolong the survival and decreased the risk of recurrence in triple-negative breast cancer[39]. Although the proportion of CD4\u0026thinsp;+\u0026thinsp;T cells has decreased, we detected that the percentage of CD3+/CD4+/CD45RO\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;central memory T cells was increased in 7D12-CAR-hIL-21-T cells group. We suspected that this CD3+/CD4+/CD45RO\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;T cells may include the increase of T helper (Th) cells, which may be a direction for subsequent research on the effect of IL-21 on CAR-T cell function. Furthermore, the modified CAR-T cells exhibited enhanced IFN-g secretion and improved anti-tumor ability when co-cultured with KYSE-30, KYSE-150 and KYSE-510 tumor cells.\u003c/p\u003e \u003cp\u003eIn vivo experiments confirmed the in vitro findings, showing effective tumor suppression and prolonged survival in KYSE-150 tumor-bearing mice treated with our CAR-T cells. To further evaluate the efficacy of the treatment, we assessed the secretion of IFN-g in the peripheral blood of 7D12-CAR-hIL-21-T cells treated mice at three different time points. While there was no significant difference in IFN-g secretion over time, the 7D12-CAR-hIL-21-T cells treated mice exhibited improved tumor suppression. Although EGFR is expressed at low levels in normal tissues, we observed no significant tissue damage in our mouse model, supporting the safety and potential of 7D12-hIL-21-CAR-T cells as a viable immunotherapy for ESCC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eThe human esophageal cancer cell lines KYSE-30, KYSE-70, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, and KYSE-510, along with the normal esophageal epithelial cell line HET-1A, were cultured in RPMI-1640 medium (GIBCO, Thermo Fisher Scientific, USA). Human T cells were maintained in X-VIVO 15 media (Lonza) supplemented with 100 IU/mL human IL-2 (PeproTech). All culture media were enriched with 10% (v/v) fetal bovine serum (FBS, Biological Industries, Israel) and 1% penicillin-streptomycin to support optimal growth conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and expansion of T cells\u003c/h2\u003e \u003cp\u003eHuman peripheral blood mononuclear cells (PBMCs) were obtained from three healthy donors and isolated by density gradient centrifugation as previously described[40]. Human T cells were then isolated from PBMC by anti-human CD3/CD28 Dynabeads (Gibco) and cultured in X-VIVO 15 (LONZA) supplemented with 5%(v/v) fetal bovine serum, 100IU/ml human IL2 (Peprotech), 100 mM β-mercaptoethanol (Sigma) and 1% penicillin-streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCAR construction\u003c/h2\u003e \u003cp\u003eThe lentiviral vectors encoding the 7D12-CAR and the 7D12-CAR-IL-21 constructs represent the second-generation CAR architecture. This design incorporates the human CD8 hinge and transmembrane domains, coupled with the intracellular co-stimulation domain of human 4-1BB and the CD3ζ signaling domain. The specificity for human EGFR was conferred by the anti-human-EGFR-VHH 7D12, as previously characterized by Rob C. Rovers et al[41]. A human CD8 signal peptide was engineered upstream of the 7D12 sequence. Untransduced (UTD) T cells were employed as a negative control within the experimental framework.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus production and T cells transduction\u003c/h2\u003e \u003cp\u003eAll lentivirus of CAR were produced by 293T cells. Simply, the 293T cells were seeded at 6x10\u003csup\u003e6\u003c/sup\u003e cells per 10-cm dish 12 hours before transfection. The transfer plasmids that contain CARs sequence, the two package plasmids pMDLg/pRRE and pRSV-Rev, and the envelope plasmid pCMV-VSV-G were transfected simultaneously into 293T cells using polyethyleneimine (PEI) transfection in a ratio of 15:7.5:6:6. The medium was replaced 10 hours later and harvested at 48 and 72 hours after transfection. Harvested lentivirus particles were first removed impurities by filtration with a 0.45\u0026micro;m membrane and then concentrated by ultracentrifugation at 10,000 x g for 16 hours. All lentiviruses are resuspended with X-VIVO culture medium and frozen at -80\u0026deg;C. Human CD3 T cells were isolated and stimulated by anti-human CD3/CD28 beads at day 0. After 48 hours of invitro stimulation, 1x10\u003csup\u003e5\u003c/sup\u003e T cells were seeded with lentivirus at a multiplicity of infection (MOI) 5 in the presence of 100\u0026micro;g/ml Synperonic\u0026reg; F 108(Sigma) as assisted transfection reagents in a 24-well plate and infected at 32℃,1200xg for 90 minutes. The transduction medium was replaced another 4 hours later and then T cells were cultured with fresh medium every two days at cell density in 3-5x10\u003csup\u003e5\u003c/sup\u003e per ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry assays\u003c/h2\u003e \u003cp\u003eFor cell membrane stain, 5x10\u003csup\u003e5\u003c/sup\u003e cells were incubated with 2.5\u0026micro;g/ml fluorescent antibodies at 4℃ for 30 min protected from light. After culture, cells were washed with 1x wash buffer (BD bioscience) and then suspended for staining the second antibody as described before. The following antibodies were used in this topic: EGFR Monoclonal Antibody(H11) (thermos-fisher MA5-13070); Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H\u0026thinsp;+\u0026thinsp;L) (Beyotime A0428); MonoRab\u0026trade; Rabbit Anti-Camelid VHH Cocktail [iFluor 488] (Genscript). The human T cells surface markers were detected using a FITC-conjugated mouse anti-humanCD3 antibody, Alexa Fluor 700-conjugated mouse anti-human CD4 antibody, Percp/cy5.5-conjugated mouse anti-human CD8 antibody, Brilliant Violet 785-conjugated mouse anti-human CD45RA antibody, Brilliant Violet 650-conjugated mouse anti-human CD45RA antibody, PE-conjugated mouse anti-human CD197 (CCR7) antibody, Brilliant Violet 421-conjugated mouse anti-human CD45RO antibody and PE-CY7-conjugated mouse anti-human CD27 antibody (all from BioLegend).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity assays\u003c/h2\u003e \u003cp\u003eThe Cytotoxicity LDH Assay Kit-WST (DOJINDO CK12) was used to detect the LDH secretion of KYSE cell lines that co-cultured with CAR-T cells or UTD T cells at different ratios (T cells: target cells\u0026thinsp;=\u0026thinsp;2:1, 4:1, 8:1, 16:1, 32:1). After 16 hours, the culture supernatants were collected and detected by using LDH release assay, human IFN-γ ELISA kit (MultiSciences 70-EK1804) and human TFN-α ELISA kit (MultiSciences 70-EK182) respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of human IL-21\u003c/h2\u003e \u003cp\u003eThe human IL-21 Uncoated ELISA Kit with plates (Invitrogen 88-8218-86) was used to measure IL-21 concentrations. Briefly, 1x106 CAR-T cells and UTD T cells were cultured in 1mL X-VIVO medium with 5%(v/v) fetal bovine serum. Cell culture supernatants were collected and assayed at 24 hours. The detected of hIL-21 was performed according to the manufacturer\u0026rsquo;s instructions of the human IL-21 Uncoated ELISA Kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBulk RNA-sequencing\u003c/h2\u003e \u003cp\u003eRNA-seq was performed by Shanghai OE Biotech Co., Ltd. Briefly, total RNA was extracted from T cells that cultured invitro including 7D12-CAR-T cells and 7D12-CAR-hIL-21-T cells using mirVana\u0026trade; miRNA ISOlation Kit, (Ambion-1561). Briefly, RNA integrity was assessed by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina, RS-122-2101), total 4 \u0026micro;g RNA was used to build library construction. Next, the libraries underwent sequencing via the Illumina HiSeq X Ten sequencing platform. These 150bp paired-end reads generated as raw data. The initial data underwent purification to obtain high-quality, clean data. Thereafter, the clean data was utilized to acquire in-depth transcriptional information following the human genome (NCBI_GRCh38.p13). GO and KEGG pathway analyses were conducted on genes meeting the criteria of \u0026plusmn;\u0026thinsp;2-fold change and corrected P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eXenograft tumor models\u003c/h2\u003e \u003cp\u003eFor the establishment of KYSE-150 xenograft tumor models, 6-week-old NOD/ SCID mice were inoculated s.c. with 5x10\u003csup\u003e6\u003c/sup\u003e KYSE-150 cells on the right flank. When the tumor size was ~\u0026thinsp;100 mm\u003csup\u003e3\u003c/sup\u003e in approximately 7 days, the mice were divided randomly into 5 groups (6 in each group), injected with 5x10\u003csup\u003e6\u003c/sup\u003e T cells dissolved in 100\u0026micro;l control PBS,100\u0026micro;l PBS including UTD T cells, 7D12-CAR-T cells or 7D12-CAR-hIL-21-T cells respectively. All of these cells were injected through the tail vein (i.v.) on day 1. Tumor size was detected every two days until the tumor size reached 100 mm3 when these mice were sacrificed. All animals cultured and treated were in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals and carried out in Laboratory Animal Center, Zhengzhou University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using Graph Pad Prism 6 and IBM SPSS statistical software (version 19.0). Student\u0026rsquo;s t-test was utilized to compare differences between two groups, while a one-way ANOVA test was used to analyze differences among multiple groups. Additionally, Kaplan\u0026ndash;Meier survival analysis was employed to assess differences in survival. Statistical significance was considered when the p-values were at or below 0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.L.Z, L.H. and Z.M.W. conceptualized the study. C.L.Z, Y.Y.L, H.R.G and D.Y.Z. performed experiments. C.L.Z, Y.P and X.Q.L. analyzed the data and generated the figures. S.S.L, L.H. and Z.M.W. interpreted data and reviewed the manuscript. C.L.Z, L.H. and Z.M.W. wrote the manuscript with input from all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was funded by research funding from the General Program of Henan Natural Science (222300420531).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAbnet CC, Arnold M, Wei WQ: \u003cstrong\u003eEpidemiology of Esophageal Squamous Cell Carcinoma\u003c/strong\u003e. \u003cem\u003eGastroenterology\u003c/em\u003e 2018, \u003cstrong\u003e154\u003c/strong\u003e(2):360\u0026ndash;373.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhu H, Ma X, Ye T, Wang H, Wang Z, Liu Q, Zhao K: \u003cstrong\u003eEsophageal cancer in China: Practice and research in the new era\u003c/strong\u003e. \u003cem\u003eInt J Cancer\u003c/em\u003e 2023, 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\u003cstrong\u003e129\u003c/strong\u003e(8):2013\u0026ndash;2024.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ESCC, EGFR, Nanobody, CAR-T cells, human IL-21","lastPublishedDoi":"10.21203/rs.3.rs-4485969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4485969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEsophageal squamous cell carcinoma (ESCC), a primary form of esophageal cancer, is characterized by poor outcomes and limited treatment options. Targeting EGFR with chimeric antigen receptor-modified T cells (EGFR CAR-T) has emerged as a promising therapeutic approach for ESCC. Utilizing nanobodies enhances the specificity of antigen recognition and has become a popular method in CAR-T cell therapy. In this study, we introduced an EGFR-specific nanobody, EGFR-VHH-7D12, into the receptor binding domain of EGFR CAR-T cells and incorporated human interleukin 21 (hIL-21) to boost the efficacy of these cells. Our findings show that EGFR-VHH-7D12-equipped CAR-T cells can accurately target and eliminate EGFR-positive esophageal cancer cells both in vitro and in animal models. The addition of hIL-21 not only increased the proliferation of CAR-T cells but also led to a higher formation of memory T cell subsets in vitro. Furthermore, the presence of hIL-21 in these CAR-T cells resulted in increased expression of IFN-gamma when cultured with various human esophageal cancer cell lines (KYSE-30, KYSE-150, and KYSE-510) in vitro. Notably, CAR-T cells expressing both EGFR-VHH-7D12 and hIL-21 showed superior anti-tumor activity in a KYSE-150 xenograft mouse model. Our results indicate that the combined expression of hIL-21 in 7D12-CAR-T cells significantly enhances their anti-tumor capabilities, making them a highly promising option for ESCC treatment.\u003c/p\u003e","manuscriptTitle":"Development of a new EGFR-VHH-CAR T-cell therapy for treatment of esophageal squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-25 11:15:05","doi":"10.21203/rs.3.rs-4485969/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"97574896-7896-4c13-bf45-c5a1937ae7b1","owner":[],"postedDate":"June 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33597676,"name":"Biological sciences/Cancer"},{"id":33597677,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2024-07-27T06:32:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-25 11:15:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4485969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4485969","identity":"rs-4485969","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}