Ligand-based adoptive T cell targeting CA125 in ovarian cancer

Ligand-based adoptive T cell targeting CA125 in ovarian cancer | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ligand-based adoptive T cell targeting CA125 in ovarian cancer Haihong Zhao, Lina Wu, Jiemin Dai, Ke Sun, Zhenguo zi, Junhua Guan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2605996/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Sep, 2023 Read the published version in Journal of Translational Medicine → Version 1 posted 3 You are reading this latest preprint version Abstract Background: Ovarian cancer (OC) is a globally malignant gynecological disease with aggressive nature, most OC cases are diagnosed at late stages and the 5-year overall survival was less than 35% after conventional treatment. Furthermore, OC patients are rarely responsive to immune-check-point inhibitor therapy, and the efficacy of CAR-T therapy was also modest due to inefficient T cell infiltration. Hence, additional approaches to improve T cell traffic in OC tumor sites need to be developed. Methods: In this report, based on the structure of the chimeric antigen receptor, we developed a novel adoptive T cell therapy with a ligand-receptor as the binding motif to improve CA125 targeting therapeutic effect against ovarian cancer. As mesothelin can naturally bind to CA125 with high affinity, the core-binding fragment of mesothelin was concatenated with 4-1BB and CD3ζ signal fragments to assemble a novel CA125-targeting chimeric receptor (CR). Meanwhile, the CAR structure targeting CA125 derived from the 4H11 antibody was also analogously constructed. CR and CAR coding RNA were electroporated into T cells to test their antitumor activity both in vitro and in vivo. Results: While CR-T or CAR-T has shown moderate activity targeting two ovarian cancer cell lines, CR and CAR co-expressed T cells had a superior killing effect than T cells expressing CR or CAR alone. Upon interaction with ovarian tumors, the activation markers and functional cytokine release abilities were also significantly increased in CR and CAR co-expressed T cells. Similarly, CR and CAR co-expressed T cells could persistently control transplanted ovarian cancer tumor growth in NOD/SCID mice and prolong the overall survival of tumor-challenged mice. Transcriptome sequencing showed that survival and cytotoxicity properties of CR and CAR co-expressed T cells were significantly altered compared with T cells expressing CR or CAR alone. Conclusion: Our work demonstrates that CA125 targeting CR and CAR can synergistically kill ovarian cancer, suggesting these two binding motifs simultaneously targeting CA125 on tumors may yield improved responses in ovarian cancer treatment. Chimeric antigen receptor (CAR) Chimeric receptor (CR) CA125 Mesothelin Ovarian cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Ovarian cancer is a fatal gynecologic disease with a top mortality rate among gynecologic malignant tumors. Due to their indolent presentation and the lack of effective screening tools, more than 75% of ovarian cancer are diagnosed at late stages with poor 5-year overall survival of less than 35 percent[1]. Chemotherapy based on cisplatin or carboplatin, the conventional treatment for ovarian cancer, is still the first-line treatment. However, tumor recurrence of advanced ovarian cancer after chemotherapy is quite common [2]. Those highly aggressive cancers are often regarded as immunologically “Cold” Tumors, a variety of Immune Checkpoint Inhibitors (ICI) and cytokine therapy are gradually entering the clinical application, but the therapeutic effect still has not been significantly improved[3]. Chimeric antigen receptor T cells (CAR-T) therapy has had a transformative clinical performance against hematological malignancies in recent years[4], which provides us with a revolutionized strategy for solid tumor treatment. Recently, several CAR-T therapies for ovarian cancer have begun to be explored, such as FOLR1[5, 6], MSLN[7], TAG-72, and CD47 [8]. However, the research progress of CAR-T for ovarian cancer has not made a breakthrough, while neurotoxicity and system side effects of CAR-T cells have been reported continuously[9, 10]. Accordingly, based on preclinical evidence, the function of CAR-T needs to have a moderate affinity with the targeting protein to prevent the dysfunction of T cells[11, 12]. Therefore, fine-tuning the affinity of CAR structure to the target can be an effective means to improve clinical response. Common CAR structure is a specific antibody scFv domain linked with immunoreceptor tyrosine-based activation motif (ITAM). However, the scFv affinity was often laboratory selected without accuracy and rationale [13]. Alternatively, it is found that partial HGF fragments can bind with MET expressed on tumor cells with high specificity [14]. Therefore, it is worth further studying whether the interaction between natural ligands and receptors could be applied to CAR design. Mesothelin and CA125 are both overexpressed in ~88% of ovarian cancer. CA125 is a highly glycosylated mucin that comprises a large cleaved and released domain and a conserved domain[15]. Moreover, 4H11 is a specific hybridoma-generated antibody targeting the conserved domain of CA125 which has already been applied to CAR-T therapy and tested in phase I clinical trial[16]. Additionally, it has been widely reported that the glycosylphosphatidylinositol-anchored glycoprotein mesothelin binds to CA125 with strong binding kinetics, facilitating peritoneal metastasis of ovarian tumors [17-20]. Glycosylated and non-glycosylated mesothelin can bind to the 156 amino acids region of membrane expressed CA125 [18]. While 64 amino acids at the N-terminal of mesothelin (region 296-359) are the minimum fragment that completes binding to CA125 [21]. In this study, we genetically fused this sequence with the 4-1BB and CD3ζ signal fragments tandemly to generate a novel structure named chimeric receptor (CR). Meanwhile, we also obtained the scFv sequence from CA125 specific antibody (4H11)[22] and constructed the CA125 targeting CAR structure as a parallel comparison. Here we aimed to systematically examine the antitumor activity of our engineered CR or CAR-expressed T cells targeting ovarian tumor cell lines, offering an alternative way for ovarian cancer treatment. Methods Mice and cell lines 6-week-old immuno-deficient NOD/SCID female mice were purchased and bred under pathogen-free conditions at the Shanghai Model organisms Center for Cancer Research. Mice were maintained in 12:12 h light: dark cycles at 60% humidity and 21–25 °C. Experiments were performed under the instruction of the requirements of the National Institutes of Health and Institutional Animal Care and Use Committee. SKOV3, OVCAR3, and U251 tumor cell lines were obtained from the ATCC cell bank with authenticated STR profiling and mycoplasma-free. Both cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS and 100 U/mL penicillin/streptomycin sulfate. All cell lines were transduced with the lentivirus of firefly luciferase-GFP (LucG) and sorted on a Bio-Rad S3e cell sorter to obtain 100% transduced populations. T cells were isolated from peripheral blood mononuclear cells (PBMCs) using ficoll density gradient centrifugation and cultured in X-VIVO 15 medium (Lonza) supplied with 100 U/mL IL-2 (R&D system), T cell activation was performed according to a previous protocol[23]. Briefly, isolated T cells were mixed with Dynabeads Human T-Activator CD3/CD28 (Gibco) in a 1:3 ratio and cultured in IL-2 containing X-VIVO 15 medium, removed dynabeads using DynaMag™-2 Grate (Gibco) after 5 days, then cultured activated T cells and supplied with IL-2 containing medium to keep T cell growth. When T cells were expanded and the cell size declined to about 350 μm3, those T cells are thought ready to be transformed or cryopreserved. Engineered T-cell production The CA125 binding sequences of CR and CAR were expatriated in Table 1. Generally, the partial mesothelin sequences and 4H11 scFv were synthesized (Sangon Biotech) under the regulation of a T7 promoter and separately cloned into the L482A RNA expression plasmid backbone. All constructs contained a CD8 hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain with 150 bp poly-A cassette. The two plasmids were in vitro transcript into RNA using capped RNA synthesis Kit (Hongene Biotech) under the manufacturer’s construction. Synthesized RNAs were allocated and saved in a -80℃ refrigerator. 1e7 prepared T cells were mixed with 5 ug CR, CAR, or 5ug CR+CAR mRNA in 100 μl opti-MEM medium (Gibco), and transferred into 0.2 cm Gene Pulser Cuvette, then the mixtures were performed electroporation using BTX ECM830 instrument under 500V, 0.7 ms[24]. After electroporation, the T cells were immediately transferred into a pre-warmed T cell culture medium and cultured in cell incubators for 18 to 24 hours. qPCR and Western blot Total RNA was extracted using RNAeasy Mini Kit (QIAGEN) and cDNA was generated using Hifair II 1st strand cDNA Synthesis kit (Yeasen Biotech). Quantitative real-time PCR was done using Hieff qPCR SYBR Green Master Mix (Yeasen Biotech). The primer for the detection of CR and CAR was shown in Table 1 Whole-cell lysates of SKOV3, OVCAR3, and U251 cell lines for western blot were generated in 200 mL RIPA buffer (Beyotime Biotech) containing protease inhibitors cocktail (Roche). Samples were incubated on ice for 10 minutes and then subjected to BCA analysis. 20 mg of total protein was used for western blot analysis. The following primary antibodies were used: anti-MUC16 antibody, and anti-β-actin antibody (Abcam). And secondary antibody was Goat anti-rabbit-IgG-HRP (Abcam). Cytotoxicity assay CR-T cells, CAR-T cells, CR+CAR-T cells, and NTD cells were washed twice with IL2-free medium, co-cultured with SKOV3-lucG, OVCAR3-lucG and U251-lucG cells at E: T ratio = 1:1 in 96-well flat-bottom plates separately, triplicate for each sample. Then placed the plate into IncuCyte S3 dynamic imaging and analysis system to detect GFP fluorescence, the change of GFP is detected every 4 hours and monitored in real-time for 72 hours. Taking the fluorescence area of 0 h 0 m per well as the standard, the killing efficiency is calculated as follows: killing efficiency = (total fluorescence area - remaining fluorescence area) / total fluorescence area × 100%. Flow cytometry The CR and CAR transfection efficiency of each T cell group was detected by staining with Fc-CA125 full-length protein (ACROBiosystem) and PE-Fc antibody (ebioscience). CA125 expression on tumor cells was detected using rabbit-anti-human CA125 antibody (Abcam) and FITC-donkey-anti-rabbit antibody (ebioscience). T cell activation markers including CD69-PE and CD25-APC (ebioscience) were detected after being co-cultured with tumor cells for 24 hours; CD1107A-PE (ebioscience) expression on T cells was detected after co-cultured with tumor cells for 4 hours with Golgi-stop solution (BD Biosciences). All T cells were gated on CD3-BV421, CD8-AF700 (ebioscience) positive population. Flow cytometric analysis was performed on Attune NxT V6 flow cytometer (ThermoFisher). Acquired data were analyzed using version ten of the FlowJo software (Tree Star). ELISA and diluted ELISpot assay For ELISA assay, 1e5 effector cells per well were stimulated with 1e5 tumor cells for 24 h, in triplicate, the IL-2 and IFNγ in the supernatant were detected by DuoSet ELISA Development Systems (R&D Systems) according to the manufacturer’s instruction. For diluted ELISpot assay, 1e3 effector cells per well were stimulated with 1e5 tumor cells for 16–20 h in duplicate or triplicate, Spots were visualized with Human IFNγ precoated ELISPOT kit (DAKEWE) according to the manufacture’s instruction; Plates were scanned and analyzed by Mabtech ASTOR ELISpot Reader. Xenograft Assay SKOV3-LucG cells were collected in healthy conditions and strained using a 100 μm filter. Cells were washed in PBS and 3e6 cells were injected in 100 μl PBS in the right flank of NDG mice. Mice were treated at day 14 with 5e6 T cells in each group administered by tail-vein injection in 100 μl PBS. Tumor progression was monitored by a Vernier caliper every two or three days and by bioluminescence signal every week using an IVIS imaging system (PerkinElmer) following intraperitoneal substrate injection of D-Luciferin (30 mg ml−1). Mice’s body weight and survival were monitored every two or three days for 30 days after adoptive T cell treatment. Bulk RNA-seq sequencing 1e6 CR T cells, CAR T cells, CR+CAR T cells, and NTD T cells were co-cultured with SKOV3 cell line respectively for 24 hours, in triplicate, then the co-cultured cells were labeled training using CD45-PE antibody (Biolegend) for 15 min at room temperature, and sorted out using EasySep™ Human PE Positive Selection Kit II (stemcell). Briefly, CD45-PE stained cells were mixed with PE selection cocktail and RapidSpheres™, thereby the CD45-PE positive cells were labeled with magnet beads, next sort out these cells with a magnet. Sorting efficiency was tested using FASC above 95%, then total RNA was extracted from sorted cells using RNAeasy Mini Kit (QIAGEN) and the RNA was detected with integrity and deep sequenced by Novogene, Beijing. Functional analyses of DEGs were performed using DAVID and KEGG for ingenuity canonical pathway enrichment. Heatmap and volcano plot of differentially expressed genes in each group and Gene set enrichment analysis (GSEA) were depicted using R (version 3.6.3). Statistical methods Analyses were performed with GraphPad Prism 8 (version 8.0.1). Data are presented as mean ± s.e.m. Data between each group were compared using a two-tailed unpaired Student’s t-test as appropriate for the type of data. Significance was considered for P < 0.05 as the following: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P 0.05 as N.S. For experiments with multiple groups, multiple comparison corrections were used as indicated in the figure legends. Results Identification of CA125-targeting CAR and CR-T cells. The construction of targeting CA125 CAR and CR elements was shown in Figure 1-A. The scFv and mesothelin fragments that can bind to CA125 coding DNA sequences (Supplementary Table 1) which are respectively connected with the CD8 signal peptide, CD8 hinge region, and CD8 transmembrane coding DNA sequence as the extracellular signal region, and the extracellular signal region was concatenated with the intracellular signal region containing 4-1BB and CD3ζ to form complete CAR and CR structures, these two structures therewith were respectively constructed into RNA expressing plasmid for in vitro transcription (Figure 1-B). Harvested CR and CAR mRNA were respectively or simultaneously electroporated into resting T cells. After 4 hours, the positive ratio of CAR and CR on the T cell surface was detected using Fc labeled CA125 full-length proteins. Flow staining showed that T cells in CAR, CR, and CR+CAR groups both can combine with CA125 protein, and the binding efficiency was over 90%, which indicated the CR and CAR-expressing T cells both can recognize CA125 without binding site competition (Figure 1-C). Notably, the MFI value of CR or CAR to CA125 was quite similar, which hints that the binding ability of mesothelin and CA125 was strong enough for cellular signal transduction[25]. We also monitored the MFI value in each group after electroporation and found that the CAR and CR were still detected for 9 days (Figure S1). To confirm that the plasmids were successively transduced into cells, we designed specific primers (Supplementary Table 1) and verified through real-time quantitative PCR to detect CR and CAR elements. Compared with the none transduced group (NTD), CR-T cells specifically expressed the mesothelin fragment, while CAR-T cells specifically expressed the 4H11 scFv fragment. In addition, the CR+CAR-T group expresses the equivalent level of mesothelin fragments and scFv fragments compared with CR or CAR-expressed T cells (Fig. 1-D). Collectively, we proved that T cells with CR and CAR were successfully constructed. CA125 targeting CAR and CR co-expressed T cells synergistically enhance tumor clearance towards ovarian cancer cells Both two ovarian cancer cell lines SKOV3 and OVCAR3 highly expressed CA125, while CA125 expression in the glioma cell line U251 was undetectable through FACS staining, which could be used as a negative control (Figure 2A, B). Additionally, we determined the expression of CA125 in ovarian cancer cells by western blotting. The expression of CA125 in OVCAR3 was slightly higher than that in SKOV3, while U251 did not express CA125 (Fig. 2C, D). To evaluate the tumor clearance ability of our engineered T cells in vitro, we first stable expressed Luciferase-GFP in three tumor cells by lentiviral transduction. Then those cells co-cultured with our engineered T cells independently, and the killing ability of different groups of T cells on tumor cells was observed through Incucyte real-time dynamic cell imaging analysis system for 72 hours, we found that the killing efficiencies of CAR-T and CR-T cells were roughly the same targeted SKOV3 cells (51.9% ± 3.28%, vs. 53.29% ±9.36%). However, For CAR and CR co-expressed T cells, the killing efficiency reached 91.46% ±4.59%; Regarding OVCAR3 cells, the killing efficiency of CAR-T cells was 73.86% ±5.51%, while that of CR-T cells was only 18.68±4.59%; while the killing efficiency of CAR and CR co-expressed T cells reached 93.57% ± 6.11%, which was significantly higher than that of other groups. Meanwhile, we did not find all of our engineered T cells have a significant killing effect on the U251 cell line (Figure 2-E). Those results demonstrated that CAR and CR co-expressed T cells targeting ovarian cancer cytotoxicity are significantly enhanced, which indicated that CR and CAR have a synergistic effect for CA125-expressed ovarian cancer. CA125 targeting CAR and CR co-expression induce superior T cell activation and cytotoxicity During T cell activation, CD69, and CD25 started to express themselves on the T cell surface[26]. Therefore, we speculated that the CAR and CR co-expressed T cells synergistically contribute to the activation of T cells. After co-cultured our engineered T cells with tumor cells for 24 hours, we detected the expression level of CD69 and CD25 on T cells. Compared with the NTD group, the CD69+CD25+ cell populations in CR or CAR-expressed T cell groups were moderately up-regulated, while the CD69+CD25+ cell population in the CR+CAR-expressed T cell group was much higher compared with CR or CAR groups. The results showed that the CR and CAR co-expressing induce more T cell activation than CAR or CR expressing alone (Fig.3 A, B). Similarly, CD107A, as the most sensitive molecular marker of T cell killing function, was widely used in the functional study of NK and CD8+ T cells [27]. After co-cultured with tumor cells for 4 hours, the expression of CD107A in each engineered CD8+ T cell was also detected with the Golgi blocker treatment[28]. The results showed that the combination of CR and CAR indeed increased the expression level of CD107A in CD8+ T cells compared with CR or CAR group (Fig. 3 C, D). However, we hardly detected any CD69, CD25, and CD107A activation after co-cultured our engineered T cell with U251 cell lines (Fig. S2). This evidence collectively demonstrated that CA125 targeting CAR and CR co-expressed T cells exhibited superior activation and cytotoxicity ability towards ovarian cancer. CA125 targeting CAR and CR co-expression significantly enhanced IL2 and IFNγ secretion in T cells. Activated T cells produce IL-2 to promote the proliferation, differentiation, and survival of T cells, and simultaneously secrete pro-inflammatory cytokine IFNγ, which converts T cells into cytotoxic T cells. The secretion levels of these two factors are closely related to the treatment efficacy of CAR-T cell therapy [29]. Therefore, after co-culturing with tumor cells for 24 hours, we simultaneously detected the secretion capacity of IL2 and IFNγ secreted by our engineered T cells in each group by ELISA (Figure.4 A, B), Consistence with activation and cytotoxicity assay, the release of IL2 and IFN-γ from CR+CAR-T group was significantly boosted compared with CR or CAR-T groups targeting both SKOV3 or OVCAR3, while co-cultured with U251 cell line, IL2 and IFNγ secretion of each group was undetectable. To validate this phenomenon, we used a diluted ELIspot experiment to detect the secretion capacity of IFNγ in each engineered T cell. Similarly, we can detect few IFNγ spots in CR or CAR-T groups, but the IFNγ spots in the CR+CAR group were significantly increased (Fig.4 C, D). The above experiments showed that when both CR and CAR were expressed, the antitumor function of T cells could be significantly enhanced. CA125 targeting CAR and CR co-expressing enhances the efficacy of T cells against ovarian cancer in vivo . To evaluate the antitumor effects of our engineered T cells in vivo , we subcutaneously implanted SKOV3-LucG tumor cells into NOD/SCID mice. Fourteen days later, mice were independently infused with our engineered T cells intravenously. Through luminescence imaging, we found tumor was gradually shrunken in CAR and CR+CAR groups, while the CR group did not show any advantage in tumor control compared with NTD group (Figure. 5A). However, the CR+CAR group had a superior tumor clearance rate compared with the CAR group on day 11 through imaging statistics (Figure. 5B). We also observed that only CAR-expressed T cells and CR+CAR-expressed T cells can effectively control SKOV3 tumor cell growth after 30 days through tumor size measuring, while tumors kept growing in mice treated with NTD or CR-expressed T cells and mice were sacrificed during 15 to 20 days (Figures. 5C). Furthermore, CR+CAR-expressed T cells treated mice show obvious survival advantages over other groups (Figure 5D). In summary, we proved that CR+CAR-expressed T cells showed prominent antitumor properties in ovarian cancer in vivo models, and increased mice survival than CR or CAR-expressed T cells alone or NTD group. The effect of CR combined with CAR on T cell signaling pathway To further specifically characterize each engineered T cell, we co-cultured CR, CAR or CR+CAR expressed T cells with SKOV3 cell line for 24 hours, respectively. After T cell separation and purification, stimulated T cells in each group were obtained for transcriptome sequencing analysis. Gene expression in each group was significantly separated by hierarchical clustering analysis of their expression profiles (Fig. 6A). Notably, we found 537 genes were up-regulated in the CR group compared with the CR+CAR group, the expression of exhaustion marker PDCD1[30] was the most obvious which indicated CR+CAR can reverse the senescence-associated secretory phenotypes. And the expression of 614 genes was down-regulated, of which the expression of IL13 was the most obvious (Fig. 6B). Compared with the CAR group with CR+CAR group, 493 genes were up-regulated and 725 genes were down-regulated in the CR+CAR group, and immune-regulator genes including IL9, IL13, BATF3 CXCL13, and several TNFRSF family members were significantly down-regulated, which associated with T cell homing and memory phenotype persistence[31-33]. We speculated that CR naturally binding CA125 can offer suitable T cell differentiation signals to promote T cell survival and persistence, while CAR stimulation can offer T cell activation signals to promote T cell cytotoxic function (Fig. 6C). Next, we performed the differential gene signaling pathways enrichment analysis, it was found that the PI3K/AKT signaling pathway, cell adhesion, and cytokine receptor pathways were significantly changed in the CR group compared with the CR+CARgroup (Figure 6D); Cell adhesion, Th17 cell differentiation and oxidative phosphorylation pathways were significantly changed between the CAR group and CR+CAR group (Fig. 6E). Gene-set enrichment analysis (GSEA) showed that glycolytic pathways and IL6/JAK/STAT3 are elevated while oxidative phosphorylation (OP) (Figure. 6F) and fatty acid metabolism (FAM) is degraded in the CAR group compared with CR+CAR group (Figure. 6G). As OP and FAM were associated with mitochondria fitness[34], we proved CR+CAR activation could synergistically promote T cell persistence and function. In conclusion, these results demonstrated that T cells in the CR+CAR group significantly altered gene-expressing profiles and signal pathways, which mainly contribute to the activation and cytotoxicity properties of T cells targeting CA125. Discussion With the rapid development of genetic engineering and the in-depth study of the molecular mechanism of T cell recognition, scientists developed chimeric antigen receptor (CAR), which endows T cells with the ability to recognize tumor antigens in an HLA-independent manner and enables them to recognize more extensive target antigens than natural T cell surface receptor[35]. . However, the treatment of solid tumors with CAR-T cells is facing multiple obstacles. On the one hand, scFv, as a synthetic antibody, leads to internalization and degradation of tumor antigen in the course of treatment, leading to treatment failure[36]; On the other hand, due to the complex tumor microenvironment and heterogeneity of solid tumors, conventional T cells cannot be activated at the tumor site, causing tumor cells to escape[37]. In ovarian cancer, mesothelin and CA125 interaction promote ovarian cancer cells and stromal tumors to metastasize, indicating that this combination has a certain tumor-promoting effect. In this study, we designed a new type of chimeric receptor T cells targeting CA125, based on the natural binding of mesothelin to CA125 in ovarian cancer patients, to reverse the role of this ligand-receptor binding activity in promoting tumor transformation. In addition, because stable expressed CAR-T may cause on-target-off tumor effects which can be life-threatening[38], we transient expressed CR and CAR into T cells as a safety consideration. Our research shows that the CA125 targeting therapeutic effect of CR-T cells alone is similar to that of CAR-T cells, but cannot eliminate tumor cells. However, CR and CAR co-expressing T cells targeting CA125 show prominent antitumor activity consisting of the expression of cell activation markers CD69 and CD25 and the expression of cell early activation marker CD107A. CR or CAR-T cells can bind to CA125, but cannot manipulate cell function in a biological environment. This may due to CR or CAR binding to CA125 is not in a stable state. The extracellular segment of CA125 contains 156 amino acid glycosylation repeat sequences, which is very easy to hydrolyze[39], making CA125 secreted into serum and body fluid, resulting in insufficient activation signals. However, when CR and CAR are co-expressed on T cells, the therapeutic effect of T cells can be significantly improved supported by IL-2 and IFNγ secretion ability, which suggests that CR-T may reduce the resistance reaction of tumor cells during the treatment process, so that CR and CAR may prolong the timing of CA125 interaction. In addition, this superior antitumor effect of CR and CAR-expressed T cells was also proved in vivo . Through transcriptome sequencing, we also proved that the combination of CR and CAR has a significant impact on the expression level of multiple immune-related genes and signal pathways during T-cell activation. Notably, the up-regulation of IL13 was most significant in CR and CAR co-expressed T cells compared with CR or CAR-only expressed T cells. IL13 was an immunoregulatory cytokine produced primarily by Th2 cells, which may benefit T cell regulation and prevent T cell overreaction [40, 41]. In addition, IL9[31], BATF3[32], and CXCR13[33], associated with T cell homing and memory phenotype persistence also up-regulated in the CR+CAR group compared to CAR only group, indicating CR interaction could reverse CAR-induced T cell overactivation, fine tunes T cells in a healthy state. Moreover, GSEA analysis also proved enhanced mitochondria fitness[42] in activated CR and CAR expressed T cells. Both pieces of evidence proved that CR was functionally beneficial for T cells. In summary, our data highlight a novel T cell engineering strategy that combines with ligand-receptor motif and antibody-antigen motif that could enhance tumor reaction and optimize T cell phenotypes, providing the translational potential for enhancing CAR T cell therapeutic efficacy in ovarian cancers. Abbreviations OC: Ovarian cancer CR: Chimeric receptor CAR: Chimeric antigen receptor ITAM: immunoreceptor tyrosine-based activation motif PBMC: peripheral blood mononuclear cells ELISA: enzyme-linked immunosorbent assay ELISpot: enzyme-linked immunospot assay Declarations Ethics approval and consent to participate The research was approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Model organisms Center Co., Ltd. Consent for publication All subjects have informed consent. Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available due to further analysis but are available from the corresponding author on reasonable request. Competing interests No potential conflicts of interest were disclosed. Funding This work was supported by the Minhang district Science and Technology Commission of Shanghai (2020MHZ026) Authors' contributions Haihong Zhao and Lina Wu contribute equally to this work. Conceptualization, Haihong Zhao; Data curation, Ke Sun; Formal analysis, Ke Sun and Zhenguo Zi; Funding acquisition, Haihong Zhao; Investigation, Lina Wu; Methodology, Lina Wu; Resources, Jiemin Dai; Software, Jiemin Dai; Supervision, Liwen Zhang; Validation, Zhenguo Zi; Visualization, Haihong Zhao; Writing – original draft, Haihong Zhao; Writing – review & editing, Junhua Guan and Liwen Zhang. Acknowledgements Not applicable References Matulonis, U.A., et al., Ovarian cancer. Nat Rev Dis Primers, 2016. 2 : p. 16061. Hennessy, B.T., R.L. Coleman, and M. Markman, Ovarian cancer. Lancet, 2009. 374 (9698): p. 1371-82. Banville, A.C., et al., Co-expression patterns of chimeric antigen receptor (CAR)-T cell target antigens in primary and recurrent ovarian cancer. Gynecol Oncol, 2021. 160 (2): p. 520-529. Neelapu, S.S., et al., Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med, 2017. 377 (26): p. 2531-2544. 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Wei, F., et al., Strength of PD-1 signaling differentially affects T-cell effector functions. Proc Natl Acad Sci U S A, 2013. 110 (27): p. E2480-9. Zhao, Y., et al., Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines. J Immunol, 2005. 174 (7): p. 4415-23. Garg, G., et al., Novel treatment option for MUC16-positive malignancies with the targeted TRAIL-based fusion protein Meso-TR3. BMC Cancer, 2014. 14 : p. 35. Caruso, A., et al., Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation. Cytometry, 1997. 27 (1): p. 71-6. Aktas, E., et al., Relationship between CD107a expression and cytotoxic activity. Cell Immunol, 2009. 254 (2): p. 149-54. Betts, M.R., et al., Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods, 2003. 281 (1-2): p. 65-78. Zi, Z., et al., B7-H3 Chimeric Antigen Receptor Redirected T Cells Target Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma. Cancers (Basel), 2020. 12 (12). Wang, T.W., et al., Blocking PD-L1-PD-1 improves senescence surveillance and ageing phenotypes. Nature, 2022. 611 (7935): p. 358-364. Liu, L., et al., Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9. Nat Commun, 2020. 11 (1): p. 5902. Ataide, M.A., et al., BATF3 programs CD8(+) T cell memory. Nat Immunol, 2020. 21 (11): p. 1397-1407. Liu, B., et al., Single-cell meta-analyses reveal responses of tumor-reactive CXCL13(+) T cells to immune-checkpoint blockade. Nat Cancer, 2022. 3 (9): p. 1123-1136. Corrado, M. and E.L. Pearce, Targeting memory T cell metabolism to improve immunity. J Clin Invest, 2022. 132 (1). Sadelain, M., I. Riviere, and R. Brentjens, Targeting tumours with genetically enhanced T lymphocytes. Nat Rev Cancer, 2003. 3 (1): p. 35-45. Camviel, N., et al., Both APRIL and antibody-fragment-based CAR T cells for myeloma induce BCMA downmodulation by trogocytosis and internalization. J Immunother Cancer, 2022. 10 (11). Yu, S., et al., Chimeric antigen receptor T cells: a novel therapy for solid tumors. J Hematol Oncol, 2017. 10 (1): p. 78. Lamers, C.H., et al., Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol Ther, 2013. 21 (4): p. 904-12. O'Brien, T.J., et al., The CA 125 gene: an extracellular superstructure dominated by repeat sequences. Tumour Biol, 2001. 22 (6): p. 348-66. Minty, A., et al., Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature, 1993. 362 (6417): p. 248-50. Proto, J.D., et al., Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation Resolution. Immunity, 2018. 49 (4): p. 666-677 e6. Wenes, M., et al., The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function. Cell Metab, 2022. 34 (5): p. 731-746 e9. Supplementary Files supplementfigure.pdf Tableinformation.xlsx Cite Share Download PDF Status: Published Journal Publication published 05 Sep, 2023 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 22 Mar, 2023 Editor assigned by journal 16 Mar, 2023 First submitted to journal 27 Feb, 2023 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-2605996","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":185751599,"identity":"3652b85b-89d2-4431-be52-83c84f1ca9dd","order_by":0,"name":"Haihong Zhao","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haihong","middleName":"","lastName":"Zhao","suffix":""},{"id":185751600,"identity":"ef0273b7-05e3-4789-8b38-a2e4dc0c6834","order_by":1,"name":"Lina Wu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Wu","suffix":""},{"id":185751601,"identity":"b8c3a189-140a-441b-86fa-a30749b60599","order_by":2,"name":"Jiemin Dai","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiemin","middleName":"","lastName":"Dai","suffix":""},{"id":185751602,"identity":"683cfd11-a51b-4fc5-91ec-d2b8842617f0","order_by":3,"name":"Ke Sun","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Sun","suffix":""},{"id":185751603,"identity":"a6900e78-3b5b-4ea9-94b6-97bfa6865cac","order_by":4,"name":"Zhenguo zi","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenguo","middleName":"","lastName":"zi","suffix":""},{"id":185751604,"identity":"2dbb0705-b2e8-4f3e-9241-5fbe22aff204","order_by":5,"name":"Junhua Guan","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junhua","middleName":"","lastName":"Guan","suffix":""},{"id":185751605,"identity":"225bd2d1-9611-411c-924d-14ea2c6520b7","order_by":6,"name":"Liwen Zhang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-6452-6568","institution":"Fudan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Liwen","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-02-20 03:52:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2605996/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2605996/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-023-04271-8","type":"published","date":"2023-09-05T15:01:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":34799246,"identity":"e4c59150-f9e6-48e7-bb93-257417fbd55b","added_by":"auto","created_at":"2023-03-24 21:34:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2783063,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction and detection of CR and CAR expressed T cells. A. The original structure of chimeric antigen receptor (CAR) and chimeric receptor (CR) targeting CA125; B. The structure of CR and CAR-expressing plasmids; C. Flow cytometry detection of the CA125 binding activity of T cells transduced with CR, CAR, and CR+CAR; D. qPCR results of mRNA expression levels of electro-transduced CR, CAR, and CR+CAR expressed T cells group after 24 hours. P \u0026gt; 0.05 labeled as N.S was considered no significance.\u003c/p\u003e","description":"","filename":"Figure151.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/30f43bc29a04e352300a4234.jpg"},{"id":34799249,"identity":"ef0569a4-d972-4c73-bb05-ce8cd8a9bcd0","added_by":"auto","created_at":"2023-03-24 21:34:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2431531,"visible":true,"origin":"","legend":"\u003cp\u003eCA125 expression in tumors and killing assay. A. Flow cytometry to detect the expression of CA125 on the surface of SKOV3, OVCAR3, and U251 cells; B. Summary of CA125 staining MFI of SKOV3, OVCAR3, and U251 cells; C. Western blot to detect the protein expression of CA125 in SKOV3, OVCAR3, and U251 cells; D. Statistic of CA125 expression detected using western blot. E. Detection of the killing ability of CR, CAR, and CR+CAR expressed T cells targeting SKOV3, OVCAR3, and U251. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, N.S as P \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure152.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/5d7fc79707429d80b1549fe5.jpg"},{"id":34799245,"identity":"59242a54-d194-45b0-8b70-c2667a2551f7","added_by":"auto","created_at":"2023-03-24 21:34:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2275408,"visible":true,"origin":"","legend":"\u003cp\u003eActivation marker detection in our engineered T cells co-cultured with tumor cells. A. Flow cytometry detection of CD69 and CD25 expression in CR, CAR, CR+CAR, and NTD groups after co-culture of T cells with ovarian cancer cells SKOV3 and OVCAR3 for 24 hours. B. Statistical chart of the proportion of CD69 and CD25 double-positive T cells in each group. C. Flow cytometry detection of CD107A expression in CR, CAR, CR+CAR, and NTD groups after co-culture of T cells with ovarian cancer cells SKOV3 and OVCAR3 for 4 hours; D. Statistical chart of CD107A positive population in T cells of each group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure153.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/d400e014d4e9a871f5722e07.jpg"},{"id":34799250,"identity":"51995cee-0068-4635-8a35-c487aaa024ba","added_by":"auto","created_at":"2023-03-24 21:34:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1794641,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine secretion of activated engineered T cells. A. ELISA detection of IL2 secretion capacity after CR, CAR, CR+CAR, and NTD T cells co-cultured with tumor cells for 24 hours; B. ELISA detection of IFN after 24 hours of co-culture between T cells and tumor cells in each group - Secretory capacity of gamma. C. Diluted ELISpot detection of IFN-γ secretion capacity after co-culture of T cells and tumor cells in each group for 24 hours; D. Statistical graph of the number of IFNγ spots in each group in ELISpot experiment. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure154.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/7de7bc0c3db7a8fba047f7c9.jpg"},{"id":34799251,"identity":"3644dc91-b8c8-4544-999a-29290623ab43","added_by":"auto","created_at":"2023-03-24 21:34:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2097142,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of engineered T cells targeting ovarian cancer cell line in vivo. (A) Representative bioluminescence images of tumor killing capacity of engineered T cells in NOD/SCID mice with SKOV3-LucG xenograft model. (B) Quantification of bioluminescence kinetics of SKOV3-LucG tumor growth in the xenograft model after T cell injection at day 11. (C) The tumor size of each group was measured via a Vernier caliper.\u0026nbsp;\u0026nbsp;(D) The Kaplan–Meier survival curve of mice in the SKOV3-LucG xenograft model. Mice were sacrificed when the tumor volume reached 2000 mm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Figure155.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/b7909ea25f9f2fab1578fca9.jpg"},{"id":34799252,"identity":"6996b566-4966-42a2-9077-f7b3a35f998e","added_by":"auto","created_at":"2023-03-24 21:34:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7290447,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional signatures and functional properties of our engineered T cells. A. Heat map of differences in gene expression in the whole transcriptome after 24 hours of co-culture of CR, CAR, CR+CAR, and NTD T cells and tumor cells. B. Volcano plots show differences in transcriptional gene expression between CR and CR+CAR group T cells and tumor cells after 24 h of co-culture. C. Volcano plot of volcanic differences in transcriptional gene expression between T cells in the CAR and CR+CAR groups and tumor cells after 24 hours of co-culture; D. KEGG pathway analysis of CR and CR+CAR group T cells expression difference gene mainly involved in the signaling pathway; E. KEGG pathway analyzes the signaling pathways in which T cells in the CAR and CR+CAR groups express differences in genes. F. Gene set enrichment analysis of IL6/JAK/STAT3 and Glycolysis signaling gene sets in CAR and CR+CAR T cells from RNA-seq, the heatmap of the top 20 up-regulated genes in the CAR group was listed in each gene set. G. Gene set enrichment analysis of Oxidative Phosphorylation and Fatty Acid Metabolism signaling gene sets in CAR and CR+CAR T cells from RNA-seq, the heatmap of the top 20 down-regulated genes in the CAR group was listed in each gene set. (n = 3 biological replicates).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/77eb646713fe60fd834ea813.jpg"},{"id":42947631,"identity":"0d93c707-2b8c-4f0d-9bcd-8920d66af512","added_by":"auto","created_at":"2023-09-11 15:10:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":986662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/767c0753-16ad-4329-8e76-8249506cc84e.pdf"},{"id":34799248,"identity":"043a8617-2aff-4589-bec6-daa57fed6e1f","added_by":"auto","created_at":"2023-03-24 21:34:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":382419,"visible":true,"origin":"","legend":"","description":"","filename":"supplementfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/ba2c31b85c14b7bdca57b1c6.pdf"},{"id":34799584,"identity":"08114256-c113-4e7f-8f44-7612b8676c56","added_by":"auto","created_at":"2023-03-24 21:42:47","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10750,"visible":true,"origin":"","legend":"","description":"","filename":"Tableinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2605996/v1/4818701fdc52d104b846f997.xlsx"}],"financialInterests":"","formattedTitle":"Ligand-based adoptive T cell targeting CA125 in ovarian cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eOvarian cancer is a fatal gynecologic disease with a top mortality rate among gynecologic malignant tumors. Due to their indolent presentation and the lack of effective screening tools, more than 75% of ovarian cancer are diagnosed at late stages with poor 5-year overall survival of less than 35 percent[1]. Chemotherapy based on cisplatin or carboplatin, the conventional treatment for ovarian cancer, is still the first-line treatment. However, tumor recurrence of advanced ovarian cancer after chemotherapy is quite common\u0026nbsp;[2]. Those highly aggressive cancers\u0026nbsp;are often regarded as immunologically \u0026ldquo;Cold\u0026rdquo; Tumors, a variety of Immune Checkpoint Inhibitors (ICI) and cytokine therapy are gradually entering the clinical application, but the therapeutic effect still has not been significantly improved[3].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChimeric antigen receptor T cells (CAR-T) therapy has had a transformative clinical performance against hematological malignancies in recent years[4], which provides us with a revolutionized strategy for solid tumor treatment. Recently, several CAR-T therapies for ovarian cancer have begun to be explored, such as FOLR1[5, 6], MSLN[7], TAG-72, and CD47\u0026nbsp;[8]. However, the research progress of CAR-T for ovarian cancer has not made a breakthrough, while neurotoxicity and system side effects of CAR-T cells have been reported continuously[9, 10]. Accordingly, based on preclinical evidence, the function of CAR-T needs to have a moderate affinity with the targeting protein to prevent the dysfunction of T cells[11, 12]. Therefore, fine-tuning the affinity of CAR structure to the target can be an effective means to improve clinical response. Common CAR structure is a specific antibody scFv domain linked with immunoreceptor tyrosine-based activation motif (ITAM). However, the scFv affinity was often laboratory selected without accuracy and rationale\u0026nbsp;[13]. Alternatively, it is found that partial HGF fragments can bind with MET expressed on tumor cells with\u0026nbsp;high specificity\u0026nbsp;[14]. Therefore, it is worth further studying whether the interaction between natural ligands and receptors could be applied to CAR design.\u003c/p\u003e\n\u003cp\u003eMesothelin and CA125 are both overexpressed in ~88% of ovarian cancer. CA125 is a highly glycosylated mucin that comprises a large cleaved and released domain and a conserved domain[15]. Moreover, 4H11 is a specific hybridoma-generated antibody targeting the conserved domain of CA125 which has already been applied to CAR-T therapy and tested in phase I clinical trial[16]. Additionally, it has been widely reported that the glycosylphosphatidylinositol-anchored glycoprotein mesothelin binds to CA125 with\u0026nbsp;strong binding kinetics, facilitating peritoneal metastasis of ovarian tumors\u0026nbsp;[17-20]. Glycosylated and non-glycosylated mesothelin can bind to the 156 amino acids region of membrane expressed CA125\u0026nbsp;[18]. While 64 amino acids at the N-terminal of mesothelin (region 296-359) are the minimum fragment that completes binding to CA125\u0026nbsp;[21]. In this study, we genetically fused this sequence with the\u0026nbsp;4-1BB and CD3\u0026zeta; signal fragments\u0026nbsp;tandemly to generate a novel structure named chimeric receptor (CR). Meanwhile, we also obtained the scFv sequence from CA125 specific antibody (4H11)[22]\u0026nbsp;and constructed the CA125 targeting CAR structure as a parallel comparison. Here we aimed to systematically examine the antitumor activity of our engineered CR or CAR-expressed T cells targeting ovarian tumor cell lines, offering an alternative way for ovarian cancer treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMice and cell lines\u003c/p\u003e\n\u003cp\u003e6-week-old immuno-deficient NOD/SCID female mice were purchased and bred under pathogen-free conditions at the Shanghai Model organisms Center for Cancer Research. Mice were maintained in 12:12\u0026thinsp;h light: dark cycles at 60% humidity and 21\u0026ndash;25 \u0026deg;C. Experiments were performed under the instruction of the requirements of the National Institutes of Health and Institutional Animal Care and Use Committee.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSKOV3, OVCAR3, and U251 tumor cell lines were obtained from the ATCC cell bank with authenticated STR profiling and mycoplasma-free. Both cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS and 100 U/mL penicillin/streptomycin sulfate. All cell lines were transduced with the lentivirus of firefly luciferase-GFP (LucG) and sorted on a Bio-Rad S3e cell sorter to obtain 100% transduced populations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eT cells were isolated from peripheral blood mononuclear cells (PBMCs) using ficoll density gradient centrifugation and cultured in X-VIVO 15 medium (Lonza) supplied with 100 U/mL IL-2 (R\u0026amp;D system), T cell activation was performed according to a previous protocol[23]. Briefly, isolated T cells were mixed with Dynabeads Human T-Activator CD3/CD28 (Gibco) in a 1:3 ratio and cultured in IL-2 containing X-VIVO 15 medium, removed dynabeads using DynaMag\u0026trade;-2 Grate (Gibco) after 5 days, then cultured activated T cells and supplied with IL-2 containing medium to keep T cell growth. When T cells were expanded and the cell size declined to about 350 \u0026mu;m3, those T cells are thought ready to be transformed or cryopreserved. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEngineered T-cell production\u003c/p\u003e\n\u003cp\u003eThe CA125 binding sequences of CR and CAR were expatriated in Table 1. Generally, the partial mesothelin sequences and 4H11 scFv were synthesized (Sangon Biotech) under the regulation of a T7 promoter and separately cloned into the L482A RNA expression plasmid backbone. All constructs contained a CD8 hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3\u0026zeta; signaling domain with 150 bp poly-A cassette. The two plasmids were in vitro transcript into RNA using capped RNA synthesis Kit (Hongene Biotech) under the manufacturer\u0026rsquo;s construction. Synthesized RNAs were allocated and saved in a -80℃ refrigerator.\u003c/p\u003e\n\u003cp\u003e1e7 prepared T cells were mixed with 5 ug CR, CAR, or 5ug CR+CAR mRNA in 100 \u0026mu;l opti-MEM medium (Gibco), and transferred into 0.2 cm Gene Pulser Cuvette, then the mixtures were performed electroporation using BTX ECM830 instrument under 500V, 0.7 ms[24]. After electroporation, the T cells were immediately transferred into a pre-warmed T cell culture medium and cultured in cell incubators for 18 to 24 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eqPCR and Western blot\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using RNAeasy Mini Kit (QIAGEN) and cDNA was generated using Hifair II 1st strand cDNA Synthesis kit (Yeasen Biotech). Quantitative real-time PCR was done using Hieff qPCR SYBR Green Master Mix (Yeasen Biotech). The primer for the detection of CR and CAR was shown in Table 1\u003c/p\u003e\n\u003cp\u003eWhole-cell lysates of SKOV3, OVCAR3, and U251 cell lines for western blot were generated in 200 mL RIPA buffer (Beyotime Biotech) containing protease inhibitors cocktail (Roche). Samples were incubated on ice for 10 minutes and then subjected to BCA analysis. 20 mg of total protein was used for western blot analysis. The following primary antibodies were used: anti-MUC16 antibody, and anti-\u0026beta;-actin antibody (Abcam). And secondary antibody was Goat anti-rabbit-IgG-HRP (Abcam).\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCytotoxicity assay\u003c/p\u003e\n\u003cp\u003eCR-T cells, CAR-T cells, CR+CAR-T cells, and NTD cells were washed twice with IL2-free medium, co-cultured with SKOV3-lucG, OVCAR3-lucG and U251-lucG cells at E: T ratio = 1:1 in 96-well flat-bottom plates separately, triplicate for each sample. Then placed the plate into IncuCyte S3 dynamic imaging and analysis system to detect GFP fluorescence, the change of GFP is detected every 4 hours and monitored in real-time for 72 hours. Taking the fluorescence area of 0 h 0 m per well as the standard, the killing efficiency is calculated as follows: killing efficiency = (total fluorescence area - remaining fluorescence area) / total fluorescence area \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003eFlow cytometry\u003c/p\u003e\n\u003cp\u003eThe CR and CAR transfection efficiency of each T cell group was detected by staining with Fc-CA125 full-length protein (ACROBiosystem) and PE-Fc antibody (ebioscience). CA125 expression on tumor cells was detected using rabbit-anti-human CA125 antibody (Abcam) and FITC-donkey-anti-rabbit antibody (ebioscience). T cell activation markers including CD69-PE and CD25-APC (ebioscience) were detected after being co-cultured with tumor cells for 24 hours; CD1107A-PE (ebioscience) expression on T cells was detected after co-cultured with tumor cells for 4 hours with Golgi-stop solution (BD Biosciences). All T cells were gated on CD3-BV421, CD8-AF700 (ebioscience) positive population. Flow cytometric analysis was performed on Attune NxT V6 flow cytometer (ThermoFisher). Acquired data were analyzed using version ten of the FlowJo software (Tree Star).\u003c/p\u003e\n\u003cp\u003eELISA and diluted ELISpot assay\u003c/p\u003e\n\u003cp\u003eFor ELISA assay, 1e5 effector cells per well were stimulated with 1e5 tumor cells for 24 h, in triplicate, the IL-2 and IFN\u0026gamma; in the supernatant were detected by DuoSet ELISA Development Systems (R\u0026amp;D Systems) according to the manufacturer\u0026rsquo;s instruction. For diluted ELISpot assay, 1e3 effector cells per well were stimulated with 1e5 tumor cells for 16\u0026ndash;20 h in duplicate or triplicate, Spots were visualized with Human IFN\u0026gamma; precoated ELISPOT kit (DAKEWE) according to the manufacture\u0026rsquo;s instruction; Plates were scanned and analyzed by Mabtech ASTOR ELISpot Reader.\u003c/p\u003e\n\u003cp\u003eXenograft Assay\u003c/p\u003e\n\u003cp\u003eSKOV3-LucG cells were collected in healthy conditions and strained using a 100 \u0026mu;m filter. Cells were washed in PBS and 3e6 cells were injected in 100 \u0026mu;l PBS in the right flank of NDG mice. Mice were treated at day 14 with 5e6 T cells in each group administered by tail-vein injection in 100 \u0026mu;l PBS. Tumor progression was monitored by a Vernier caliper every two or three days and by bioluminescence signal every week using an IVIS imaging system (PerkinElmer) following intraperitoneal substrate injection of D-Luciferin (30\u0026thinsp;mg ml\u0026minus;1). Mice\u0026rsquo;s body weight and survival were monitored every two or three days for 30 days after adoptive T cell treatment.\u003c/p\u003e\n\u003cp\u003eBulk RNA-seq sequencing\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; 1e6 CR T cells, CAR T cells, CR+CAR T cells, and NTD T cells were co-cultured with SKOV3 cell line respectively for 24 hours, in triplicate, then the co-cultured cells were labeled training using CD45-PE antibody (Biolegend) for 15 min at room temperature, and sorted out using EasySep\u0026trade; Human PE Positive Selection Kit II (stemcell). Briefly, CD45-PE stained cells were mixed with PE selection cocktail and RapidSpheres\u0026trade;, thereby the CD45-PE positive cells were labeled with magnet beads, next sort out these cells with a magnet. Sorting efficiency was tested using FASC above 95%, then total RNA was extracted from sorted cells using RNAeasy Mini Kit (QIAGEN) and the RNA was detected with integrity and deep sequenced by Novogene, Beijing. Functional analyses of DEGs were performed using DAVID and KEGG for ingenuity canonical pathway enrichment. Heatmap and volcano plot of differentially expressed genes in each group and Gene set enrichment analysis (GSEA) were depicted using R (version 3.6.3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatistical methods\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalyses were performed with GraphPad Prism 8 (version 8.0.1). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.e.m. Data between each group were compared using a two-tailed unpaired Student\u0026rsquo;s t-test as appropriate for the type of data. Significance was considered for P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the following: *P\u0026thinsp;\u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, no significance was considered for P \u0026gt; 0.05 as N.S. For experiments with multiple groups, multiple comparison corrections were used as indicated in the figure legends.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of CA125-targeting CAR and CR-T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe construction of targeting CA125 CAR and CR elements was shown in Figure 1-A. The scFv and mesothelin fragments that can bind to CA125 coding DNA sequences (Supplementary Table 1) which are respectively connected with the CD8 signal peptide, CD8 hinge region, and CD8 transmembrane coding DNA sequence as the extracellular signal region, and the extracellular signal region was concatenated with the intracellular signal region containing 4-1BB and CD3\u0026zeta; to form complete CAR and CR structures, these two structures therewith were respectively constructed into RNA expressing plasmid for \u003cem\u003ein vitro\u003c/em\u003e transcription (Figure 1-B). Harvested CR and CAR mRNA were respectively or simultaneously electroporated into resting T cells. After 4 hours, the positive ratio of CAR and CR on the T cell surface was detected using Fc labeled CA125 full-length proteins. Flow staining showed that T cells in CAR, CR, and CR+CAR groups both can combine with CA125 protein, and the binding efficiency was over 90%, which indicated the CR and CAR-expressing T cells both can recognize CA125 without binding site competition (Figure 1-C). Notably, the MFI value of CR or CAR to CA125 was quite similar, which hints that the binding ability of mesothelin and CA125 was strong enough for cellular signal transduction[25]. We also monitored the MFI value in each group after electroporation and found that the CAR and CR were still detected for 9 days (Figure S1). To confirm that the plasmids were successively transduced into cells, we designed specific primers (Supplementary Table 1) and verified through real-time quantitative PCR to detect CR and CAR elements. Compared with the none transduced group (NTD), CR-T cells specifically expressed the mesothelin fragment, while CAR-T cells specifically expressed the 4H11 scFv fragment. In addition, the CR+CAR-T group expresses the equivalent level of mesothelin fragments and scFv fragments compared with CR or CAR-expressed T cells (Fig. 1-D). Collectively, we proved that T cells with CR and CAR were successfully constructed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCA125 targeting CAR and CR co-expressed T cells synergistically enhance tumor clearance towards ovarian cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth two ovarian cancer cell lines SKOV3 and OVCAR3 highly expressed CA125, while CA125 expression in the glioma cell line U251 was undetectable through FACS staining, which could be used as a negative control (Figure 2A, B). Additionally, we determined the expression of CA125 in ovarian cancer cells by western blotting. The expression of CA125 in OVCAR3 was slightly higher than that in SKOV3, while U251 did not express CA125 (Fig. 2C, D). To evaluate the tumor clearance ability of our engineered T cells in vitro, we first stable expressed Luciferase-GFP in three tumor cells by lentiviral transduction. Then those cells co-cultured with our engineered T cells independently, and the killing ability of different groups of T cells on tumor cells was observed through Incucyte real-time dynamic cell imaging analysis system for 72 hours, we found that the killing efficiencies of CAR-T and CR-T cells were roughly the same targeted SKOV3 cells (51.9% \u0026plusmn; 3.28%, vs. 53.29% \u0026plusmn;9.36%). However, For CAR and CR co-expressed T cells, the killing efficiency reached 91.46% \u0026plusmn;4.59%; Regarding OVCAR3 cells, the killing efficiency of CAR-T cells was 73.86% \u0026plusmn;5.51%, while that of CR-T cells was only 18.68\u0026plusmn;4.59%; while the killing efficiency of CAR and CR co-expressed T cells reached 93.57% \u0026plusmn; 6.11%, which was significantly higher than that of other groups. Meanwhile, we did not find all of our engineered T cells have a significant killing effect on the U251 cell line\u0026nbsp;(Figure 2-E). Those results demonstrated that CAR and CR co-expressed T cells targeting ovarian cancer cytotoxicity are significantly enhanced, which indicated that CR and CAR have a synergistic effect for CA125-expressed ovarian cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCA125 targeting CAR and CR co-expression induce superior T cell activation and cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring T cell activation, CD69, and CD25 started to express themselves on the T cell surface[26]. Therefore, we speculated that the CAR and CR co-expressed T cells synergistically contribute to the activation of T cells. After co-cultured our engineered T cells with tumor cells for 24 hours, we detected the expression level of CD69 and CD25 on T cells. Compared with the NTD group, the CD69+CD25+ cell populations in CR or CAR-expressed T cell groups were moderately up-regulated, while the CD69+CD25+ cell population in the CR+CAR-expressed T cell group was much higher compared with CR or CAR groups. The results showed that the CR and CAR co-expressing induce more T cell activation than CAR or CR expressing alone (Fig.3 A, B). Similarly, CD107A, as the most sensitive molecular marker of T cell killing function, was widely used in the functional study of NK and CD8+ T cells\u0026nbsp;[27]. After co-cultured with tumor cells for 4 hours, the expression of CD107A in each engineered CD8+ T cell was also detected with the Golgi blocker treatment[28]. The results showed that the combination of CR and CAR indeed increased the expression level of CD107A in CD8+ T cells compared with CR or CAR group (Fig. 3 C, D). However, we hardly detected any CD69, CD25, and CD107A activation after co-cultured our engineered T cell with U251 cell lines (Fig. S2). This evidence collectively demonstrated that CA125 targeting CAR and CR co-expressed T cells exhibited superior activation and cytotoxicity ability towards ovarian cancer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCA125 targeting CAR and CR co-expression significantly enhanced IL2 and IFN\u0026gamma; secretion in T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivated T cells produce IL-2 to promote the proliferation, differentiation, and survival of T cells, and simultaneously secrete pro-inflammatory cytokine IFN\u0026gamma;, which converts T cells into cytotoxic T cells. The secretion levels of these two factors are closely related to the treatment efficacy of CAR-T cell therapy\u0026nbsp;[29]. Therefore, after co-culturing with tumor cells for 24 hours, we simultaneously detected the secretion capacity of IL2 and IFN\u0026gamma; secreted by our engineered T cells in each group by ELISA (Figure.4 A, B), Consistence with activation and cytotoxicity assay, the release of IL2 and IFN-\u0026gamma; from CR+CAR-T group was significantly boosted compared with CR or CAR-T groups targeting both SKOV3 or OVCAR3, while co-cultured with U251 cell line, IL2 and IFN\u0026gamma; secretion of each group was undetectable. To validate this phenomenon, we used a diluted ELIspot experiment to detect the secretion capacity of IFN\u0026gamma; in each engineered T cell. Similarly, we can detect few IFN\u0026gamma; spots in CR or CAR-T groups, but the IFN\u0026gamma; spots in the CR+CAR group were significantly increased (Fig.4 C, D). The above experiments showed that when both CR and CAR were expressed, the antitumor function of T cells could be significantly enhanced.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCA125 targeting CAR and CR co-expressing enhances the efficacy of T cells against ovarian cancer \u003cem\u003ein vivo\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the antitumor effects of our engineered T cells \u003cem\u003ein vivo\u003c/em\u003e, we subcutaneously implanted SKOV3-LucG tumor cells into NOD/SCID mice. Fourteen days later, mice were independently infused with our engineered T cells intravenously. Through luminescence imaging, we found tumor was gradually shrunken in CAR and CR+CAR groups, while the CR group did not show any advantage in tumor control compared with NTD group (Figure. 5A). However, the CR+CAR group had a superior tumor clearance rate compared with the CAR group on day 11 through imaging statistics (Figure. 5B). We also observed that only CAR-expressed T cells and CR+CAR-expressed T cells can effectively control SKOV3 tumor cell growth after 30 days through tumor size measuring, while tumors kept growing in mice treated with NTD or CR-expressed T cells and mice were sacrificed during 15 to 20 days (Figures. 5C). Furthermore, CR+CAR-expressed T cells treated mice show obvious survival advantages over other groups (Figure 5D). In summary, we proved that CR+CAR-expressed T cells showed prominent antitumor properties in ovarian cancer\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e models, and increased mice survival than CR or CAR-expressed T cells alone or NTD group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of CR combined with CAR on T cell signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further specifically characterize each engineered T cell, we co-cultured CR, CAR or CR+CAR expressed T cells with SKOV3 cell line for 24 hours, respectively. After T cell separation and purification, stimulated T cells in each group were obtained for transcriptome sequencing analysis. Gene expression in each group was significantly separated by hierarchical clustering analysis of their expression profiles (Fig. 6A). Notably, we found 537 genes were up-regulated in the CR group compared with the CR+CAR group, the expression of exhaustion marker PDCD1[30]\u0026nbsp;was the most obvious which indicated CR+CAR can reverse the senescence-associated secretory phenotypes. And the expression of 614 genes was down-regulated, of which the expression of IL13 was the most obvious (Fig. 6B). Compared with the CAR group with CR+CAR group, 493 genes were up-regulated and 725 genes were down-regulated in the CR+CAR group, and immune-regulator genes including IL9, IL13, BATF3 CXCL13, and several TNFRSF family members were significantly down-regulated, which associated with T cell homing and memory phenotype persistence[31-33]. We speculated that CR naturally binding CA125 can offer suitable T cell differentiation signals to promote T cell survival and persistence, while CAR stimulation can offer T cell activation signals to promote T cell cytotoxic function (Fig. 6C). Next, we performed the differential gene signaling pathways enrichment analysis, it was found that the PI3K/AKT signaling pathway, cell adhesion, and cytokine receptor pathways were significantly changed in the CR group compared with the CR+CARgroup (Figure 6D); Cell adhesion, Th17 cell differentiation and oxidative phosphorylation pathways were significantly changed between the CAR group and CR+CAR group (Fig. 6E). Gene-set enrichment analysis (GSEA) showed that glycolytic pathways and IL6/JAK/STAT3 are elevated while oxidative phosphorylation (OP) (Figure. 6F) and fatty acid metabolism (FAM) is degraded in the CAR group compared with CR+CAR group (Figure. 6G). As OP and FAM were associated with mitochondria fitness[34], we proved CR+CAR activation could synergistically promote T cell persistence and function. In conclusion, these results demonstrated that T cells in the CR+CAR group significantly altered gene-expressing profiles and signal pathways, which mainly contribute to the activation and cytotoxicity properties of T cells targeting CA125.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith the rapid development of genetic engineering and the in-depth study of the molecular mechanism of T cell recognition, scientists developed chimeric antigen receptor (CAR), which endows T cells with the ability to recognize tumor antigens in an HLA-independent manner and enables them to recognize more extensive target antigens than natural T cell surface receptor[35]. .\u0026nbsp;However, the\u0026nbsp;treatment of solid tumors with CAR-T cells is facing\u0026nbsp;multiple obstacles.\u0026nbsp;On the one hand, scFv, as a synthetic antibody, leads to internalization and degradation of tumor antigen in the course of treatment, leading to treatment failure[36]; On the other hand, due to the complex tumor microenvironment and heterogeneity of solid tumors, conventional T cells cannot be activated at the tumor site, causing tumor cells to escape[37]. In ovarian cancer, mesothelin and CA125 interaction promote ovarian cancer cells and stromal tumors to metastasize, indicating that this combination has a certain tumor-promoting effect. In this study, we designed a new type of chimeric receptor T cells targeting CA125, based on the natural binding of mesothelin to CA125 in ovarian cancer patients, to reverse the role of this ligand-receptor binding activity in promoting tumor transformation. In addition, because stable expressed CAR-T may cause on-target-off tumor effects which can be life-threatening[38], we transient expressed CR and CAR into T cells as a safety consideration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur research shows that the CA125 targeting therapeutic effect of CR-T cells alone is similar to that of CAR-T cells, but cannot eliminate tumor cells. However, CR and CAR co-expressing T cells targeting CA125 show prominent antitumor activity consisting of the expression of cell activation markers CD69 and CD25 and the expression of cell early activation marker CD107A. CR or CAR-T cells can bind to CA125, but cannot manipulate cell function in a biological environment. This may due to CR or CAR binding to CA125 is not in a stable state. The extracellular segment of CA125 contains 156 amino acid glycosylation repeat sequences, which is very easy to hydrolyze[39], making CA125 secreted into serum and body fluid, resulting in insufficient activation signals. However, when CR and CAR are co-expressed on T cells, the therapeutic effect of T cells can be significantly improved supported by IL-2 and IFN\u0026gamma; secretion ability, which suggests that CR-T may reduce the resistance reaction of tumor cells during the treatment process, so that CR and CAR may prolong the timing of CA125 interaction. In addition, this superior antitumor effect of CR and CAR-expressed T cells was also proved \u003cem\u003ein vivo\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThrough transcriptome sequencing, we also proved that the combination of CR and CAR has a significant impact on the expression level of multiple immune-related genes and signal pathways during T-cell activation. Notably, the up-regulation of IL13 was most significant in CR and CAR co-expressed T cells compared with CR or CAR-only expressed T cells. IL13 was an immunoregulatory cytokine produced primarily by Th2 cells, which may benefit T cell regulation and prevent T cell overreaction\u0026nbsp;[40, 41]. In addition, IL9[31], BATF3[32], and \u0026nbsp;CXCR13[33], associated with T cell homing and memory phenotype persistence also up-regulated in the CR+CAR group compared to CAR only group, indicating CR interaction could reverse CAR-induced T cell overactivation, fine tunes T cells in a healthy state. Moreover, GSEA analysis also proved enhanced mitochondria fitness[42]\u0026nbsp;in activated CR and CAR expressed T cells. Both pieces of evidence proved that CR was functionally beneficial for T cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, our data highlight a novel T cell engineering strategy that combines with ligand-receptor motif and antibody-antigen motif that could enhance tumor reaction and optimize T cell phenotypes, providing the translational potential for enhancing CAR T cell therapeutic efficacy in ovarian cancers.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOC: Ovarian cancer\u003c/p\u003e\n\u003cp\u003eCR: Chimeric receptor\u003c/p\u003e\n\u003cp\u003eCAR: Chimeric antigen receptor\u003c/p\u003e\n\u003cp\u003eITAM: immunoreceptor tyrosine-based activation motif\u003c/p\u003e\n\u003cp\u003ePBMC: peripheral blood mononuclear cells\u003c/p\u003e\n\u003cp\u003eELISA: enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eELISpot: enzyme-linked immunospot assay\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Model organisms Center Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll subjects have informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are not publicly available due to further analysis but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflicts of interest were disclosed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the\u0026nbsp;Minhang district Science and Technology Commission of Shanghai (2020MHZ026)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaihong Zhao and Lina Wu contribute equally to this work.\u003c/p\u003e\n\u003cp\u003eConceptualization, Haihong Zhao; Data curation, Ke Sun; Formal analysis, Ke Sun and Zhenguo Zi; Funding acquisition, Haihong Zhao; Investigation, Lina Wu; Methodology, Lina Wu; Resources, Jiemin Dai; Software, Jiemin Dai; Supervision, Liwen Zhang; Validation, Zhenguo Zi; Visualization, Haihong Zhao; Writing \u0026ndash; original draft, Haihong Zhao; Writing \u0026ndash; review \u0026amp; editing, Junhua Guan and Liwen Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMatulonis, U.A., et al., \u003cem\u003eOvarian cancer.\u003c/em\u003e Nat Rev Dis Primers, 2016. \u003cstrong\u003e2\u003c/strong\u003e: p. 16061.\u003c/li\u003e\n\u003cli\u003eHennessy, B.T., R.L. 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Furthermore, OC patients are rarely responsive to immune-check-point inhibitor therapy, and the efficacy of CAR-T therapy was also modest due to inefficient T cell infiltration. Hence, additional approaches to improve T cell traffic in OC tumor sites need to be developed.\u003c/p\u003e\n\u003cp\u003eMethods: In this report, based on the structure of the chimeric antigen receptor, we developed a novel adoptive T cell therapy with a ligand-receptor as the binding motif to improve CA125 targeting therapeutic effect against ovarian cancer. As mesothelin can naturally bind to CA125 with high affinity, the core-binding fragment of mesothelin was concatenated with 4-1BB and CD3ζ signal fragments to assemble a novel CA125-targeting chimeric receptor (CR). Meanwhile, the CAR structure targeting CA125 derived from the 4H11 antibody was also analogously constructed. CR and CAR coding RNA were electroporated into T cells to test their antitumor activity both in vitro and in vivo.\u003c/p\u003e\n\u003cp\u003eResults: While CR-T or CAR-T has shown moderate activity targeting two ovarian cancer cell lines, CR and CAR co-expressed T cells had a superior killing effect than T cells expressing CR or CAR alone. Upon interaction with ovarian tumors, the activation markers and functional cytokine release abilities were also significantly increased in CR and CAR co-expressed T cells. Similarly, CR and CAR co-expressed T cells could persistently control transplanted ovarian cancer tumor growth in NOD/SCID mice and prolong the overall survival of tumor-challenged mice. Transcriptome sequencing showed that survival and cytotoxicity properties of CR and CAR co-expressed T cells were significantly altered compared with T cells expressing CR or CAR alone.\u003c/p\u003e\n\u003cp\u003eConclusion: Our work demonstrates that CA125 targeting CR and CAR can synergistically kill ovarian cancer, suggesting these two binding motifs simultaneously targeting CA125 on tumors may yield improved responses in ovarian cancer treatment.\u003c/p\u003e","manuscriptTitle":"Ligand-based adoptive T cell targeting CA125 in ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-24 21:34:42","doi":"10.21203/rs.3.rs-2605996/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-03-23T00:18:54+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-16T10:04:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2023-02-27T20:36:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89febca2-df45-4fec-8490-dcfdd3464e71","owner":[],"postedDate":"March 24th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-09-11T15:08:18+00:00","versionOfRecord":{"articleIdentity":"rs-2605996","link":"https://doi.org/10.1186/s12967-023-04271-8","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2023-09-05 15:01:44","publishedOnDateReadable":"September 5th, 2023"},"versionCreatedAt":"2023-03-24 21:34:42","video":"","vorDoi":"10.1186/s12967-023-04271-8","vorDoiUrl":"https://doi.org/10.1186/s12967-023-04271-8","workflowStages":[]},"version":"v1","identity":"rs-2605996","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2605996","identity":"rs-2605996","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}