A multifunctional T-cell engager containing CD80 enhances prostate cancer treatment

A multifunctional T-cell engager containing CD80 enhances prostate cancer treatment | 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 Research Article A multifunctional T-cell engager containing CD80 enhances prostate cancer treatment Disen Nie, Yao Jiang, Hui Li, Keying Zhang, Zhengxuan Li, Tong Lu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5391004/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 15 You are reading this latest preprint version Abstract Background: T-cell engagers (TCEs) are the most examined bispecific antibodies that recognize and target effector T cells to generate immune synapses against tumor cells, thereby resulting in T-cell activation and tumor killing. However, the inhibitory influence of the tumor microenvironment on TCEs' functionality has impeded their utilization in solid tumor therapies. To circumvent this limitation, we enhanced bispecific prostate-specific membrane antigen (PSMA)/CD3 TCEs by incorporating the extracellular segment of CD80 to overcome T-cell inhibition in prostate cancer by delivering a second activation signal to T cells, designating this trifunctional antibody as TriTE-N13. Methods： Using gene editing and eukaryotic expression techniques, we developed the fully humanized fusion antibody TriTE-N13. In vitro , we assessed its effects on T-cell activation, proliferation, and cytotoxicity, and tested the T-cell targeting cytotoxicity function of TriTE-N13 against PSMA + tumor cells in both cell co-culture models and tumor cell spheroid models. Furthermore, in humanized immune-reconstituted mouse models, we evaluated the in vivo efficacy and safety of TriTE-N13 against prostate cancer xenografts. Results: In vitro , TriTE-N13 can notably activate human T cells and exhibits excellent binding activity to human PSMA and CD3 antigens. Meanwhile, TriTE-N13 can mediate T-cell-induced cytotoxicity against PSMA-positive prostate cancer cells. In vivo , we demonstrated that TriTE-N13 significantly reduced tumor volume compared to bispecific TCEs when treating initially large-sized tumors. Conclusions: Our data suggest that the incorporation of CD80 as a second pathway activator significantly enhances the solid tumor-killing effect of TCEs, thereby positioning TriTE-N13 as a promising immunotherapy candidate for the treatment of advanced prostate cancer. T cell engager CD80 prostate cancer tri-specific antibodies PSMA/CD3 antigens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Prostate cancer is the second leading cause of cancer-related deaths in males, after lung cancer, in terms of the number of new cases per year. 1 Early prostate cancer is mainly treated through surgical resection and hormone deprivation. However, effective treatments to prevent the continuous progression of metastatic castration-resistant prostate cancer (mCRPC) are lacking, necessitating the urgent development of new therapeutic strategies. Emerging immunotherapies include tumor vaccines, chimeric antigen receptor T cells, cytokine injections, immune checkpoint inhibitors, and bispecific antibodies (BsAbs). 2 Immune checkpoint inhibitors exhibit potent and durable therapeutic effects in the clinical treatment of some types of tumors. 3,4 However, clinical trials examining the potential of these immune checkpoint inhibitors in patients with mCRPC revealed no significant objective response rates. 5 Sipuleucel-T is an innovative therapy for mCRPC that centers on utilizing the patient's autologous immune cells. These cells are activated in vitro by the action of PA2024 (a PAP-GM-CSF fusion protein), before being reintroduced into the patient’s body. This therapy functions by activating T cells to recognize and attack cancer cells that express PAP. 6 The mechanism lies in the effective triggering of the dual signaling activation pathway of T cells, including the binding of TCR/CD3 to the major histocompatibility complex (MHC) peptide complex (Signal 1) and involvement of co-stimulatory molecules such as CD28 (Signal 2) (Figure 1a), ensuring the full activation of T cells. Compared to immune checkpoint inhibitors, Sipuleucel-T has demonstrated superior efficacy by directly targeting tumor-specific antigens and has generated interest in exploring novel immunotherapies that are more efficient and universal. A new treatment approach that comprehensively activates T cells while avoiding highly personalized manufacturing processes, akin to Sipuleucel-T treatment, is warranted. CD3 BsAbs bridge the gap between T cells and tumor cells by targeting CD3 and tumor antigens. This connection is generated owing to the high affinity of antibodies, causing direct T-cell activation by replacing signal 1. Treating patients who develop resistance to PD-1 inhibitors with BsAbs could enhance their sensitivity to the PD-1 inhibitors. 7 Accordingly, BsAbs may have a unique mechanism of action for T-cell activation. Prostate-specific membrane antigen (PSMA) is a cell surface membrane protein frequently overexpressed in prostate cancer and often associated with androgen-independent prostate cancer and secondary metastatic lesions. 8 Preclinical studies have demonstrated the potential of targeting PSMA as a prostate cancer antigen, detailing various PSMAxCD3 bispecific formats in xenogeneic mouse models. 9-12 PSMAxCD3 BsAbs reportedly exert considerable antitumor effects against initially small-sized tumors; however, they are ineffective against initially large-sized tumors, 13 thereby warranting further improvements in the antitumor ability of BsAbs against mCRPC. Methods for activating signal 2 are increasingly employed to enhance the effectiveness of immunotherapy. Compared with PSMAxCD3 alone, 4-1BB co-stimulation, combined with PSMAxCD3, enhances CD8 + T-cell infiltration, prolongs T-cell activation, and increases proliferation in larger tumors. 13 A class of BsAbs that mimics signal 2 by bridging the tumor antigen to the co-stimulatory CD28 receptor on T cells can substantially improve the effect of CD3 BsAbs. 14 CD80, a CD28 ligand, plays a crucial role in T-cell activation. Physiologically, CD80 interacts with PD-L1 in cis on primary activated dendritic cells, interfering with PD-L1/PD-1 binding and subsequently abrogating PD-1 function in T cells. 15 Certain tumor cells lack or under-express CD80 molecules on their surface, and upregulating CD80 expression in these tumor cells by drug action promotes antibody-mediated T-cell killing. 16 Based on the current evidence, we considered adding a second signal based on CD3 BsAbs. We incorporated the CD80 function into BsAbs because CD80 activates CD28 and binds to PD-L1. Therefore, we constructed a fusion protein containing scFvs targeting PSMA and CD3, together with the extracellular segment of CD80. This antibody exerted a favorable therapeutic effect on prostate tumors in a preclinical experimental study. Materials and methods 2-1 Construction of expression vectors The scFv-targeting PSMA was retrieved from a large yeast-displayed human single-chain antibody library. 17 ScFv-targeting CD3 and CD80 extracellular segment sequences were screened and obtained from the NCBI public database. 18 All fragment sequences were codon-optimized (Kingsley Bioscience and Technology Co. Ltd, China) for efficient expression in HEK293F cells. The scFv (PSMA) was ligated to the scFv (CD3) via a flexible linker 3 (G 4 S) using overlap PCR. The Fc segment of IgG1 was point-mutated (N297A) using the same technique. 19 Expression sequences were constructed and inserted into the mammalian expression vector pCMV3-C-Myc (Sino Biological, China). The full sequence information of antibodies was listed in Table S1. 2-2 Antibody expression and purification The plasmid was transiently transfected into HEK293F cells using FreeStyle™ MAX Reagent (Thermo, USA) according to the kit instructions. The cells were cultured in shaker suspension (8% CO 2 , 37℃, 125rpm) using FreeStyle™ 293 Expression Medium (Gibco, USA). After 7 days, the cell culture medium was collected and centrifuged at 3500rpm for 10 min. The supernatant was mixed with an equal amount of phosphate-buffered saline (PBS) and filtered using a 0.45-μm membrane (Millipore, USA). The filtrate was purified using a Protein A column (Beyotime, China) with an ÄKTA protein purifier (GE, USA) to obtain the target antibody. The control antibody without the Fc segment structure was purified using a nickel ion chromatography column (Beyotime, China). The antibody concentration was determined using a NanoDrop2000 (Thermo Fisher Scientific, USA) microspectrophotometer after desalting and concentration; the antibody was frozen at −80°C after dispensing. 2-3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB) Purified antibodies were separated by 12% SDS-PAGE under reducing conditions, followed by Coomassie Brilliant Blue staining of the gels and detection of the signals using a Chemiluminescent Imaging System (Bio-Rad ChemiDoc XRS+, USA). Under the same electrophoresis conditions, proteins were transferred to PVDF membranes for WB. After blocking with 5% BSA, membranes were incubated overnight at 4°C with anti-His antibody (Proteintech, China) or anti-human Fc antibody (Thermo Fisher Scientific, USA), followed by incubation with anti-mouse secondary antibody/HRP conjugate (Thermo Fisher Scientific, USA) for 2 hours at room temperature. Finally, the membranes were treated with an ultrasensitive luminescent agent (Solarbio, China). Signals were detected and analyzed by Image Lab software. 2-4 Cells and culture conditions Human prostate cancer cell lines C4-2, 22Rv1, and PC-3(PSMA - )/(PSMA + ) were cultured in a RPMI-1640 (Servicebio, China) medium with 10% fetal bovine serum (FBS) (Gibco, USA). Jurkat cells and human peripheral blood mononuclear cells (PBMCs) were cultured in a RPMI-1640 medium with 15% FBS. All cells were cultured at 37°C and under 5% CO 2 . PC-3(PSMA + ) and 22Rv1-L were cell lines overexpressing the target gene (PSMA/Luciferase). With the approval of the Medical Ethics Committee of Xijing Hospital in Xi'an (KY20203128-1), peripheral blood samples were collected and PBMCs were isolated using Ficoll density gradient centrifugation. 2-5 Detecting antibody binding activity C4-2/22Rv1/PC-3(PSMA + /PSMA - ) cells were washed twice with PBS at 4°C, and the cell concentration was adjusted to 1×10 5 cells/200 µL. Antibodies of each type were added at 10 nmol/L, and cells were co-incubated on ice for 30 min. After two PBS washes and incubation with anti-IgG-Fc fluorescent antibody (Abcam, UK) for 30 min in the dark, followed by one PBS wash, the binding activity of each antibody to the PSMA was determined by detecting the fluorescence intensity using a flow cytometer (Agilent NovoCyte, China). The binding activities to the CD3 antigen in Jurkat and human T cells were determined using the same method. TriTE-N13 was incubated with C4-2/Jurkat cells at a final concentration of 0.001–10 nmol/L (doubling dilution), and the average fluorescence intensity of C4-2/Jurkat cells was detected using a flow cytometry. The equilibrium dissociation constants (KD values) of TriTE-N13 were calculated using GraphPad Prism V8.4.3. Table S2 provides detailed information regarding the commercial antibodies used. 2-6 Determining lymphocyte activation by antibodies To detect antibody-mediated T-cell activation in peripheral blood mononuclear cells (PBMCs) without tumor cells, PBMCs were cultured in 48-well plates (Thermo Fisher Scientific, USA). Different concentrations of the target antibody were added to each experimental group. At 48/60/72 h, an anti-CD25/CD69/CD3 (Thermo Fisher Scientific, USA) fluorescent antibody was used for an incubation step, and the T-cell activation level was then detected using flow cytometry. 2-7 In vitro detection of antibody-mediated killing activity C4-2/22Rv1/PC-3(PSMA + /PSMA - ) cells were selected and added to 48-well plates at 1×10 4 cells/well. Freshly extracted PBMCs were added according to the target efficiency ratio of E:T=4:1 (T is the initial cell number at the time of model establishment) after 24 h of incubation. Different concentrations (0.028/0.28/1.4/2.8/7/ 14 nmol/L) of the target antibody were added to each experimental grouping (NC/PCFC/CDFC/N2/N13) and incubated at 37°C under 5% CO 2 . The culture system was processed at 48/60/72 h according to the kit instructions (Servicebio Human IFN-gamma/IL-2/IL-6 ELISA Kit, China) to perform enzyme-linked immunosorbent assays (ELISA) of corresponding cytokines. The tumor cells were subjected to calcein/propidium iodide (PI) staining (Beyotime C2015M, China); the cell status was examined and photographed using a fluorescence microscope (Olympus FV3000, Japan). ImageJ software (V0.5.7) was used to analyze the results. 2-8 Determining antibody function in a three-dimensional (3D) tumor cell sphere model C4-2/22Rv1 cells were added to the prepared 96-well plates at 2×10 3 cells/well in 150 μL of Dulbecco's Modified Eagle Medium (DMEM) (Servicebio, China) with 10% FBS, centrifuged at 1500 ×g for 10 min, and cultured under routine conditions; the solution was changed after 3 days, and treatment experiments were initiated after 5–7 days. Freshly extracted PBMCs were resuspended in DMEM with 10% FBS and added to the well plate at the ratio of E:T=6:1. The corresponding antibody (7 nmol/L) was added according to the treatment group (NC/PBMC/PBMC+N2/PBMC+N13) and incubated at 24-h intervals using the EVOS M5000 live cell imager system (Thermo Fisher Scientific, USA) to observe the status of the tumor spheres and capture images (4×). After 3 days, the supernatant in culture wells was collected and aspirated to detect the cytokine levels using an ELISA kit. 2-9 Establishing in vivo treatment of the transplantation tumor model in immunoreconstructed mice Humanized immune reconstitution was performed using severe combined immunodeficient mice (NOD/ShiLtJPrkdc em26Cd52 Il2rg em26Cd22 /Gpt; NCG). Male NCG mice (4-5 weeks old) were purchased from the GemPharmatech Co., Ltd (Jiangsu, China). All mice weighed 20 ± 2 g and were housed in the SPF barrier facility of the Fourth Military Medical University (Xi’an, China). All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of FMMU (Protocol No. 20220705) and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (2011). Human PBMCs (2×10 7 ) were transplanted into immuno-reconstructed NCG mice via the tail vein. The construction was deemed successful when the proportion of hCD45 + T cells in mouse blood exceeded 25% (Figure S2). PBS-resuspended 22Rv1-L and (1×10 7 ) tumor cells were transplanted by subcutaneous injection on the right side of the mice; tumor volume was calculated as [length×width2]/2. Treatment experiments were initiated on day 7 after transplantation. The mouse models were randomly divided into four groups (NC/PBMC/N2/N13), and PBS/BiTE-N2/TriTE-N13 (bacterial filtration treatment, dose: 1 mg/kg) was injected through the tail vein once every 3 days. Tumor volume and mouse body weight were recorded every 3 days; the experiments were terminated when the tumor volume of the control group exceeded 2000 mm 3 . Mice were anesthetized using isoflurane mixed with oxygen, and the tumor was removed by blunt dissection through a 6–8-mm longitudinal incision. After suturing the wound, the mice were euthanized under anesthesia. The tumor tissues were formalin-fixed and paraffin-embedded, sectioned, and stained using a fluorescent antibody against human CD8/CD45. The sections were scanned using an inverted phase-contrast fluorescence microscope (OLYMPUS, IX71, Japan) at a magnification of 20×. The images were processed using Olympus FV31S-SW (V2.4.1.198). The culture medium was collected to determine the changes in human-derived cytokine levels in the serum using an ELISA kit. The heart, liver, kidney, lungs, and spleen were collected, fixed, sectioned, and stained with hematoxylin and eosin (HE); any damage to the organs and tissues was assessed. 2-10 Statistical analysis Data are presented as means ± standard deviation (SD). Statistically significant differences were determined using specific tests in GraphPad Prism V8.4.3. Statistical analysis was performed using two-way analysis of variance (ANOVA) with a post hoc Tukey’s test for multiple comparisons. P<0.05 was considered statistically significant. Results 3-1 Generating and characterizing the tri-specific antibody TriTE-N13 We initially analyzed CD80 expression in various tumors using The Cancer Genome Atlas (TCGA) data. 20 CD80 expression was markedly higher in immunotherapy-sensitive tumors, such as kidney cancer, than in the adjacent tissues (Figure 1b). However, in prostate cancer, CD80 expression was not increased in tumor tissues when compared with that in adjacent tissues (Figure 1c). Furthermore, CD80 expression remained the most unaltered when compared with other highly expressed signaling pathway molecules involved in T cell activation in prostate cancer (Figure 1d). Collectively, these data indicated that signal 2 involving CD80 cannot fully activate T cells in prostate cancer. Accordingly, we designed a bispecific antibody comprising four parts: two connected scFvs targeting CD3 and PSMA, an Fc segment with the BiTE-N297A mutation, and an extracellular group of CD80 fused to the C terminal of Fc (Figure 1e). To verify the effectiveness of this bispecific antibody, we designed control antibodies with different structures (Figure S1), including N1: PSMA(scFv)-CD3(scFv), BiTE-N2: PSMA-CD3-Fc (Figure 1f), PSMA(scFv)-Fc, and CD3(scFv)-Fc. The constructs were expressed and purified in 293F cells. TriTE-N13 is a fully human fusion antibody that effectively avoids anti-drug antibody (ADA) production during treatment. The molecular weight of TriTE-N13 is ~120 kDa (Figure 1g, h). Mass spectrometry analysis revealed that the target protein contained all the peptides from various functional regions, revealing that the antibody structure was identical and complete as designed (Table S3). 3-2 TriTE-N13 exhibits excellent binding activity on target cells Three prostate cancer cell lines (C4-2/PC-3(PSMA - )/PC-3(PSMA + )) were used to detect the binding activity of antibodies to PSMA antigens. Jurkat and T cells sorted from human PBMCs were used to detect the CD3 binding activity. TriTE-N13 bound to PSMA and CD3 with high affinity (Figure 2a, b), and the binding activity of TriTE-N13 was comparable to that of PSMA-Fc and CD3-Fc. The binding activity of TriTE-N13 was comparable to that of BiTE-N2, indicating that the CD80 segment did not influence the binding activity of TriTE-N13 (Figure 2c). 3-3 TriTE-N13 effectively activates T cells in vitro The TriTE-N13-mediated activation effect was tested on T cells treated with TriTE-N13 or BiTE-N2 (Method S4). T cells treated with the TriTE-N13 antibody alone exhibited more pronounced proliferation after 4 days of culture, with only minimal proliferation observed upon treatment with BiTE-N2, the control antibody (Figure S3). CD25/CD69 expression on CD3 + T cells was used as an indicator of T-cell activation. Treatment with TriTE-N13 activated ~20% of CD3 + T cells. These results indicated that anti-CD3 and CD80 antibody components activate T cells in the absence of tumor cells (Figure 2d, e). We subsequently determined whether stronger activation was achieved when TriTE-N13 was added to the co-culture of T cells and PSMA-positive tumor cells. TriTE-N13 increased the T-cell activation intensity by 60% in a dose-dependent manner in the presence of PSMA-positive tumor cells (Figure 2f, g, i, j). However, the TriTE-N13-mediated activation effect on T cells was weak in the co-culture of PSMA-negative PC3 and T cells, indicating that T-cell activation by TriTE-N13 was antigen-specific (Figure 2h, k). 3-4 TriTE-N13 induces T-cell killing of PSMA + tumor cells in vitro Next, we examined whether TriTE-N13 mediates the killing effect of T cells and used PI and calcein to indicate dead and live cells, respectively (Figure 3a). Compared with PSMA-negative PC-3 cells, PSMA-positive cells had a higher proportion of apoptotic cells after 60 h. TriTE-N13 and BiTE-N2 mediated a strong killing effect on all PSMA + tumor cells, which was gradually enhanced with the extension of co-culture time. Both the flow cytometry and CCK8 (Method S5) assay results revealed that TriTE-N13 exerted a more robust killing effect than BiTE-N2, with an average maximum killing rate of 80%. No significant cytotoxicity was observed in PSMA-negative PC-3 cells under the same interventions (Figure 3b, c). We further detected the interleukin (IL)-2, IL-6, and interferon (IFN)-γ levels in the culture supernatant and found that levels of all three cytokines were elevated in the TriTE-N13 and BiTE-N2 groups. TriTE-N13 effectively induced the release of these cytokines at low concentrations (Figure 3d). TriTE-N13 stimulated T-cell activation faster than BiTE-N2, as evidenced by the early detection of CD107a and TIM3 expression levels on CD8 + T cells (Figure 3e). 3-5 TriTE-N13 induces significant T-cytotoxic effects in the PSMA + tumor cell sphere model Although cell co-culture experiments at the two-dimensional (2D) level in vitro demonstrated that TriTE-N13 effectively mediated the killing effect of T cells on tumor cell lines, the pavement-like arrangement of tumor cell lines did not mimic the 3D environment of solid tumors. We established a 3D model of globular aggregated tumor cell clusters and examined the ability of TriTE-N13 to induce T cell-mediated killing activity to restore the therapeutic environment in which the spatial structure of tumors impedes T cells from generating killing effects. After the C4-2/22Rv1 cell line was successfully selected to establish the tumor cell spherical aggregation cluster model (on day 4), sufficient amounts of antibody and fresh human PBMCs were added according to the grouping for co-culture. Changes in the cell sphere morphology were analyzed to determine the antitumor effects under different treatment conditions. TriTE-N13 induced a more substantial human PBMC killing effect on tumor cell spheres than BiTE-N2. Tumor cell spheres in the TriTE-N13 group were smaller than those in the BiTE-N2 group (Figure 4a, b). The culture supernatant of the TriTE-N13 group had higher levels of various cytokines at 48 h than the culture supernatants of other control groups (Figure 4c), indicating that TriTE-N13 was more effective than BiTE-N2 in tumor models with a 3D structure. However, the BiTE-N2-mediated tumor-killing effect in the 3D model was less than that in the conventional 2D model. 3-6 TriTE-N13 effectively inhibits prostate tumor growth in a transplanted tumor model In vivo assays were performed to determine the antitumor function of the target antibody by establishing a xenograft tumor model in NCG mice with a reconstructed humanized immune system. At an initial treatment volume of <50 mm 3 , BiTE-N2 and TriTE-N13 significantly suppressed tumor growth (Figure 5a, b). Moreover, the tumor-suppressive effect of TriTE-N13 was superior to that of BiTE-N2 when the initial treatment volume of transplanted tumors was large (volume ≈100 mm 3 ). Upon discontinuation, the tumors in the BiTE-N2 group exhibited accelerated growth, whereas those in the TriTE-N13 group demonstrated sustained and effective suppression of tumor growth. This contrast further underscores the advantage of TriTE-N13 in maintaining therapeutic efficacy. (Figure 5c-f). The fluorescent staining (CD8/CD45) of tumor specimens and flow cytometry analysis of tumor tissue cell suspensions revealed that both the TriTE-N13 and BiTE-N2 groups exhibited a similar yet significant increase in lymphocyte infiltration when compared with the PBMC group (Figure 5g, h). However, the TriTE-N13 group showed the marked presence of infiltrating effector T-cells with elevated PD-1 expression, demonstrating the superior ability of TriTE-N13 to induce more effective T-cell activation (Figure 5i). To further validate the superiority of TriTE-N13 over BiTE-N2 in the large tumor volume model, we increased the initial treatment volume (~150 mm 3 ), revealing that TriTE-N13 retained the more superior tumor suppression effect when compared with BiTE-N2 (Figure 5k-m). 3-7 TriTE-N13 did not cause excessive systemic toxic reactions We focused on whether the target antibody exerted an overactivating effect on T cells in vivo , given that TriTE-N13 comprised CD80 components that activate the second pathway, and in vitro experiments revealed that TriTE-N13 activated T cells in human PBMCs. Weight changes in mice were recorded during treatment, revealing no significant inter-group differences (weight loss in the non-intervention group at the later stage may be related to the excessive tumor load) (Figure S4). Examination of hematoxylin-eosin stained sections of key organs at the end of the experiment found no significant differences in liver and kidney tissue damage among examined groups (Figure S5). ELISA was performed to detect serum levels of IL6/IFN-r in mice 21 days after initiating treatment, detecting no significant difference among examined groups. (Figure 5j). Discussion The excellent performance of TCEs in hematological oncology has not been observed in solid tumors; this is mainly attributed to the complex microenvironment surrounding solid tumors and the “off-target effect,” which cannot be well resolved by TCEs in solid tissues. The prevailing strategy involves combining different immunotherapies with TCEs to achieve T-cell retargeting while increasing T-cell activation levels. 21 Although a combination of different therapies can elicit better efficacy than TCEs alone, the use of more drugs increases the possibility of off-target effects. 22 Therefore, we designed and produced a tri-specific TCEs containing a functional group of CD80 (N13) that adds CD80 to therapeutic TCEs with a PSMA-CD3 structure, aiming to increase the CD80 exposure in synapses when TCEs induces artificial synapse formation and enhance effector T-cell activation by TCEs via the CD28–CD80 pathway, thereby improving the efficacy of immunotherapy on prostate tumors. TriTE-N13 exhibited excellent binding activity to both PSMA + and CD3 + cells and effectively activated T cells in PBMCs to produce a dose-dependent killing effect on PSMA + prostate tumor cells in vitro . We observed similar cytotoxic effects in an in vitro tumor cell sphere model. Using a xenograft tumor model of immuno-reconstructed mice, we showed that TriTE-N13 could effectively suppress tumor growth. Furthermore, TriTE-N13 and BiTE-N2 increased T-cell infiltration in the tumor, although PD-1 expression on intratumoral T cells was elevated in the TriTE-N13 group when compared with that in other groups, suggesting that TriTE-N13 could more efficiently activate immune cell infiltration. The suppressive tumor microenvironment of solid tumors requires that immunotherapy fully activates the immune system. 23 However, it is important to reconcile enhancing the immune activation level to increase efficacy while reducing the activation level to decrease side effects. TriTE-N13 is designed to reinforce the T-cell activation level by increasing CD80 exposure at the immune synapse. In our in vitro co-culture experiments, TriTE-N13 elicited T-cell activation at a lower concentration level than BiTE-N2; TriTE-N13 mediated higher levels of cytokine release in the tumor cell sphere model, with more pronounced tumor sphere suppression. Although TriTE-N13 activated T cells prior to immune synapse formation, the activated T cells possess PSMA targeting properties because of surface enrichment with TriTE-N13 (carrying the PSMA antigenic recognition site). In vivo , TriTE-N13 did not induce severe side effects when compared with BiTE-N2 while exerting a potent tumor-suppressive effect. The next stage of our research will focus on determining whether CD80 in TriTE-N13 mediates CD28 co-stimulatory signaling while further regulating the immune response via the PD-1/CTLA-4 pathway. Declarations Acknowledgements #Y. Jiang, H. Li contributed equally to this work. This study was supported by the National Natural Science Foundation of China (No. 82220108004; 82173204; 82203633), the Innovation Capability Support Program of Shaanxi (2023-CX-TD-72; 2021TD-39; 2020PT-021), and the Natural Science Basic Research Program of Shaanxi (2022JZ-62). Disclosure statement The authors have declared that no competing interest exists. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024 May-Jun;74(3):229-263. Apetoh L, Ladoire S, Coukos G, et al. Combining immunotherapy and anticancer agents: the right path to achieve cancer cure? Ann Oncol. 2015 Sep;26(9):1813-1823. Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. 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Freeman GJ, Disteche CM, Gribben JG, et al. The gene for B7, a costimulatory signal for T-cell activation, maps to chromosomal region 3q13.3-3q21. Blood. 1992 Jan 15;79(2):489-94. Treffers LW, van Houdt M, Bruggeman CW, et al. FcγRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils. Front Immunol. 2019 Jan 30; 9:3124. The results shown in the article are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. Belmontes B, Sawant DV, Zhong W, et al. Immunotherapy combinations overcome resistance to bispecific T cell engager treatment in T cell-cold solid tumors. Sci Transl Med. 2021 Aug 25;13(608): eabd1524. Goebeler ME, Bargou RC. T cell-engaging therapies - BiTEs and beyond. Nat Rev Clin Oncol. 2020 Jul;17(7):418-434. Tanaka A, Sakaguchi S. Regulatory T cells in cancer immunotherapy. Cell Res. 2017 Jan;27(1):109-118. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 10 Apr, 2025 Reviews received at journal 09 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviews received at journal 03 Apr, 2025 Reviews received at journal 03 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 10 Jan, 2025 Reviewers agreed at journal 17 Nov, 2024 Reviewers invited by journal 15 Nov, 2024 Editor assigned by journal 05 Nov, 2024 Submission checks completed at journal 05 Nov, 2024 First submitted to journal 04 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-5391004","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":379114314,"identity":"b4990ab3-3927-4d50-ab67-113b01b6e1d5","order_by":0,"name":"Disen Nie","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Disen","middleName":"","lastName":"Nie","suffix":""},{"id":379114315,"identity":"55f98bae-9b11-4ee0-bfdc-4c7cd6bee08b","order_by":1,"name":"Yao Jiang","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Jiang","suffix":""},{"id":379114316,"identity":"8db51180-bb94-4b7a-8fda-9a69a999efb4","order_by":2,"name":"Hui Li","email":"","orcid":"","institution":"Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Li","suffix":""},{"id":379114317,"identity":"d00e596a-052e-47a6-8f53-d738fa2246a1","order_by":3,"name":"Keying Zhang","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Keying","middleName":"","lastName":"Zhang","suffix":""},{"id":379114318,"identity":"19827f3c-44a2-4ec9-9203-170309526bb9","order_by":4,"name":"Zhengxuan Li","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhengxuan","middleName":"","lastName":"Li","suffix":""},{"id":379114319,"identity":"6277cc0c-bb12-4fad-a1e1-22984a10ec29","order_by":5,"name":"Tong Lu","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Lu","suffix":""},{"id":379114320,"identity":"787062b7-d34f-44cc-b435-0991e4d32342","order_by":6,"name":"Yu Li","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Li","suffix":""},{"id":379114321,"identity":"59c19fac-e402-43ae-8d28-c4270bb5f4b6","order_by":7,"name":"Donghui Han","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Donghui","middleName":"","lastName":"Han","suffix":""},{"id":379114322,"identity":"8647df07-a2a9-4e85-afc7-b43c361a22d8","order_by":8,"name":"Changhong Shi","email":"","orcid":"","institution":"Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changhong","middleName":"","lastName":"Shi","suffix":""},{"id":379114323,"identity":"8d9fe784-1ef6-4c4c-b485-e82d9b5b6986","order_by":9,"name":"Nianzeng Xing","email":"","orcid":"","institution":"National Cancer Center, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Nianzeng","middleName":"","lastName":"Xing","suffix":""},{"id":379114324,"identity":"f520f8d4-febe-41f8-a001-83f974f2cb06","order_by":10,"name":"Fa Yang","email":"","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fa","middleName":"","lastName":"Yang","suffix":""},{"id":379114325,"identity":"c42cf308-6d9a-4214-802a-6d23898f7653","order_by":11,"name":"Weihong Wen","email":"","orcid":"","institution":"Northwestern Polytechnical University","correspondingAuthor":false,"prefix":"","firstName":"Weihong","middleName":"","lastName":"Wen","suffix":""},{"id":379114326,"identity":"6d91cbfa-4e0a-41cb-ad02-36be5fa544ca","order_by":12,"name":"Weijun Qin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACZhBhwCbH3oDgEqfFmOcA0VqgILGHaC18x3mPSXwo4EvvETudJsFQYZ3YwH72AF4tkof50iRnGLDl9kjnbpNgOJOe2MCTl4BXi8FhHrPbPEAt+0FaGNsOJzZI8BgQ1vLHgC2dB6zlH7FagCGWANHSQIQWycM85j97DNgMgX7ZbJFwLN24jScHvxa+82eMDX78OSYPtGXjjQ811rL97Gfwa2E4ACaPQTgJQMyGXz1cSw1BdaNgFIyCUTCCAQBa0z83xukuCgAAAABJRU5ErkJggg==","orcid":"","institution":"Xijing Hospital, Fourth Military Medical University","correspondingAuthor":true,"prefix":"","firstName":"Weijun","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2024-11-04 23:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5391004/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5391004/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-025-04121-0","type":"published","date":"2025-07-23T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69267156,"identity":"fa4ebe19-0025-4fe1-b5fd-386380a29798","added_by":"auto","created_at":"2024-11-18 14:45:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1683572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and characterization of the tri-specific antibody TriTE-N13\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Major pathways of tumor cell-induced T cell activation; (b,c) Comparison of the expression of each activation molecule of the second pathway in kidney and prostate cancers in cancer and paracancerous tissues; (d) Comparison of CD80 expression in cancer and paracancerous tissues of different solid tumors; (e) The structure and mechanism of action of the PSMA/CD3/CD80 tri-specific TCEs (TriTE-N13); (f) The structure and mechanism of action of the PSMA/CD3 bispecific TCEs (BiTE-N2); (g) Western blot (anti-His/anti-Hum IgG-Fc) to determine the expression of each type of antibody; (h) Reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine purified TriTE-N13/ BiTE-N2. PSMA, prostate-specific membrane antigen; TCEs, T-cell engagers. N1: PSMA-CD3; N2: BiTE-N2; N13: TriTE-N13.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5391004/v1/84dd1811a79ba9576adb18ef.png"},{"id":69267155,"identity":"30373ec3-7f97-445e-8eef-6fc847cdd9e8","added_by":"auto","created_at":"2024-11-18 14:45:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1688315,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTriTE-N13 binds well to target cells and effectively activates T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Affinity analysis of TriTE-N13 with PSMA\u003csup\u003e+\u003c/sup\u003e cells (K\u003csub\u003eD\u003c/sub\u003e=2.3nM); (b) Affinity analysis of TriTE-N13 with CD3\u003csup\u003e+\u003c/sup\u003e cells (K\u003csub\u003eD\u003c/sub\u003e=1.4nM); (c) Flow cytometric analysis of the binding activity of each antibody type to Jurkat cells, human-derived T cells, and human prostate cancer cell line (22Rv1/C4-2/PC-3); (d,e) T-cell activation in human-derived PBMCs by each type of antibody: serial dilutions of TriTE-N13 and control antibodies were added to PBMCs, and flow cytometric assay was performed to detect CD25/CD69 expression on CD3\u003csup\u003e+\u003c/sup\u003e cells (48h); (f-k) Co-culture of C4-2/PC-3(PSMA\u003csup\u003e+\u003c/sup\u003e)/ PC-3(PSMA\u003csup\u003e-\u003c/sup\u003e) and PBMC: serial dilutions of TriTE-N13 and control antibodies were added to the system; (f-h) Flow cytometric assay for CD69 expression on CD3\u003csup\u003e+\u003c/sup\u003e cells (48 h); (i-k) Flow cytometric assay for CD25 expression on CD3\u003csup\u003e+\u003c/sup\u003e cells (48 h). Data are presented as the mean± standard deviation (SD) (n=3; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001); NC: PBS; PCFC: PSMA(scFv)-Fc; CDFC: CD3(scFv)-Fc; N2: BiTE-N2; N13: TriTE-N13.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5391004/v1/f097a3f2caa04abebae8689c.png"},{"id":69267158,"identity":"b712152f-01a2-4928-92a3-581c077ec1e5","added_by":"auto","created_at":"2024-11-18 14:45:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8104346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTriTE-N13 mediates substantial tumor killing by T cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(a,b,d) Human prostate cancer cell lines were co-cultured with human-derived PBMCs (E:T=4:1) at 37°C: (a) Calcein/propidium iodide staining was performed on C4-2/PC-3(PSMA\u003csup\u003e+\u003c/sup\u003e)/ PC-3(PSMA\u003csup\u003e-\u003c/sup\u003e) after adding PBS/BiTE-N2/TriTE-N13 (7 nmol/L) for 60 h of co-culture, and the live cell number within the fixed field (100 cells) was enumerated under a fluorescence microscope (duplicate wells: n=3, 2 fields of view per well); (b) C4-2/22Rv1/PC-3(PSMA\u003csup\u003e+\u003c/sup\u003e)/PC-3(PSMA\u003csup\u003e-\u003c/sup\u003e) was stained with CFSE and co-cultured with PBMCs spiked with PBS/BiTE-N2/TriTE-N13 (7 nmol/L), and the live cells proportion was detected by flow cytometric assay at each time point (24/48/72 h); (c) Human prostate cancer cell lines were co-cultured with human T cells (E:T=2:1) by adding various types of antibodies for 48 h, followed by the addition of CCK8 dye to detect antibody-mediated cytotoxicity. (d) Serial dilutions of each antibody type were added to the C4-2 co-culture system, with supernatants collected after 48 h of incubation, and levels of interleukin (IL)-2/IL-6/interferon (IFN)-α released were detected using ELISA; (e) Flow cytometry was performed to detect the expression of TIM-3/CD107a induced by each antibody type (7 nmol/L) to CD8\u003csup\u003e+\u003c/sup\u003e T cells in the C4-2 culture system. Data are presented as the mean± standard deviation (SD) (n=3; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001). CSFE: carboxyfluorescein succinimidyl ester. NC: PBS; PCFC: PSMA(scFv)-Fc; CDFC: CD3(scFv)-Fc; N2: BiTE-N2; N13: TriTE-N13.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5391004/v1/d82380c292f0f28d38b1be7c.png"},{"id":69267755,"identity":"eafcf5fa-6767-4b86-ac2b-e4bfccf0f21e","added_by":"auto","created_at":"2024-11-18 14:53:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2218405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTriTE-N13 induces significant T-cytotoxic effects in the PSMA\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e tumor cell sphere model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC4-2/22Rv1 cell spheres were co-cultured with human-derived PBMCs (E:T=6:1) at 37°C for 4 days, and PBS/BiTE-N2/TriTE-N13 (7 nmol/L) was added to groups in the culture system: (a) images were taken daily to capture morphological changes in cell spheres (scale bar in the figure: 500 μm); (b) image software analyzed changes in the cell sphere horizontal surface area to assess T cell toxicity effects; (c) culture supernatants were subjected to ELISA to detect levels of interleukin (IL)-2/IL-6/interferon (IFN)-α. Data are presented as the mean± standard deviation (SD) (n=3; *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 and ****p\u0026lt;0.0001). Rate of volumetric change: (V\u003csub\u003e3d\u003c/sub\u003e-V\u003csub\u003e0d\u003c/sub\u003e)/V\u003csub\u003e3d\u003c/sub\u003e). NC: PBS; PBMC: PBMCs+PBS; N2: PBMCs+(BiTE-N2); N13: PBMCs+ (TriTE-N13).\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5391004/v1/75db5baaa105b43755282f91.png"},{"id":69267159,"identity":"30456395-5b96-4181-8148-0442c600a840","added_by":"auto","created_at":"2024-11-18 14:45:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5065386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTriTE-N13 effectively suppresses prostate tumor growth in a xenograft tumor model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a,b) Phase 1 \u003cem\u003ein vivo\u003c/em\u003e experiments: (a) Immuno-reconstructed NCG mice were transplanted with 22Rv1-L cells subcutaneously, and PBS, BiTE-N2 (1 mg/kg), and TriTE-N13 (1 mg/kg) were injected into the tail vein on day 4 post-transplantation at a frequency of 1 injection/3 days; (b) growth curves of the 22Rv1 transplanted tumors in the 1st treatment; (c-e) Phase 2 \u003cem\u003ein vivo\u003c/em\u003e experiments: (c) NCG mice were immuno-reconstructed on day 3 after subcutaneous transplantation of 22Rv1-L cells, and antibodies were injected into the tail vein on day 7 after reconstruction with PBS, BiTE-N2 (1 mg/kg), and TriTE-N13 (1 mg/kg) at a frequency of 1 injection/3 days for a total of 4 antibody injections; (d) images of transplanted tumors at the end of the 2nd treatment; (e) growth curves of the 22Rv1 transplanted tumors in the 2nd treatment; (f) \u003cem\u003ein vivo\u003c/em\u003e bioluminescence imaging to monitor the growth of the transplanted tumor; (g) immunofluorescence staining of tissue sections to detect CD8\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003e+\u003c/sup\u003e lymphocyte infiltration in the transplanted tumor body; (h) flow cytometric assay to detect the proportion of CD8\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003e+\u003c/sup\u003e lymphocytes in the single-cell suspension of the transplanted tumor; (i) flow cytometric assay to detect the proportion of CD8\u003csup\u003e+\u003c/sup\u003e cell surface PD-1/CTLA-4 expression; (j) serum ELISA to detect (Hum) interleukin (IL)-6/interferon (IFN)-α levels in peripheral blood of mice after treatment (21d). (k-m) Phase 3 \u003cem\u003ein vivo\u003c/em\u003e experiments: (k) other conditions remained unchanged, and the number of initially transplanted 22Rv1-L cells was increased for a total of 6 antibody injections; (l) images of transplanted tumors at the end of the 3rd treatment; (m) growth curves of the 22Rv1 transplanted tumors in the 3rd treatment. Data are presented as the mean± standard deviation (SD) (Phase 1: n=3; Phase 2: n=5; Phase 3: n=4; *p\u0026lt; 0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 and ****p\u0026lt;0.0001). NC group in \u003cem\u003ein vivo\u003c/em\u003e experiments: unreconstructed NCG mice were modeled and administered PBS injection; PBMC: PBS; N2: BiTE-N2; N13: TriTE-N13.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5391004/v1/8021acaffe6c3af176490d55.png"},{"id":87756692,"identity":"22144b5b-4caf-4143-a799-1b62da851727","added_by":"auto","created_at":"2025-07-28 16:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18614673,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5391004/v1/500158f2-891b-44fa-ad04-eb18e1e2f142.pdf"},{"id":69267160,"identity":"44e3417f-bbbc-473d-8d42-86f7c700cb78","added_by":"auto","created_at":"2024-11-18 14:45:25","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6450853,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5391004/v1/e8c1852d107d511025e05317.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A multifunctional T-cell engager containing CD80 enhances prostate cancer treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer is the second leading cause of cancer-related deaths in males, after lung cancer, in terms of the number of new cases per year.\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eEarly prostate cancer is mainly treated through surgical resection and hormone deprivation. However, effective treatments to prevent the continuous progression of metastatic castration-resistant prostate cancer (mCRPC) are lacking, necessitating the urgent development of new therapeutic strategies.\u003c/p\u003e\n\u003cp\u003eEmerging immunotherapies include tumor vaccines, chimeric antigen receptor T cells, cytokine injections, immune checkpoint inhibitors, and bispecific antibodies (BsAbs).\u003csup\u003e2\u003c/sup\u003e Immune checkpoint inhibitors exhibit potent and durable therapeutic effects in the clinical treatment of some types of tumors.\u003csup\u003e3,4\u003c/sup\u003e However, clinical trials examining the potential of these immune checkpoint inhibitors in patients with mCRPC revealed no significant objective response rates.\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSipuleucel-T is an innovative therapy for mCRPC that centers on utilizing the patient\u0026apos;s autologous immune cells. These cells are activated \u003cem\u003ein vitro\u003c/em\u003e by the action of PA2024 (a PAP-GM-CSF fusion protein), before being reintroduced into the patient\u0026rsquo;s body. This therapy functions by activating T cells to recognize and attack cancer cells that express PAP.\u003csup\u003e6\u003c/sup\u003e The mechanism lies in the effective triggering of the dual signaling activation pathway of T cells, including the binding of TCR/CD3 to the major histocompatibility complex (MHC) peptide complex (Signal 1) and involvement of co-stimulatory molecules such as CD28 (Signal 2) (Figure 1a), ensuring the full activation of T cells. Compared to immune checkpoint inhibitors, Sipuleucel-T has demonstrated superior efficacy by directly targeting tumor-specific antigens and has generated interest in exploring novel immunotherapies that are more efficient and universal. A new treatment approach that comprehensively activates T cells while avoiding highly personalized manufacturing processes, akin to Sipuleucel-T treatment, is warranted.\u003c/p\u003e\n\u003cp\u003eCD3 BsAbs bridge the gap between T cells and tumor cells by targeting CD3 and tumor antigens. This connection is generated owing to the high affinity of antibodies, causing direct T-cell activation by replacing signal 1.\u0026nbsp;Treating patients who develop resistance to PD-1 inhibitors with BsAbs could enhance their sensitivity to the PD-1 inhibitors.\u003csup\u003e7\u003c/sup\u003e Accordingly, BsAbs may have a unique mechanism of action for T-cell activation. Prostate-specific membrane antigen\u0026nbsp;(PSMA) is a cell surface membrane protein frequently overexpressed in prostate cancer and often associated with androgen-independent prostate cancer and secondary metastatic lesions.\u003csup\u003e8\u003c/sup\u003e Preclinical studies have demonstrated the potential of targeting PSMA as a prostate cancer antigen, detailing various PSMAxCD3 bispecific formats in xenogeneic mouse models.\u003csup\u003e9-12\u003c/sup\u003e PSMAxCD3 BsAbs reportedly exert considerable antitumor effects against initially small-sized tumors; however, they are ineffective against initially large-sized tumors,\u003csup\u003e13\u003c/sup\u003e thereby warranting further improvements in the antitumor ability of BsAbs against mCRPC.\u003c/p\u003e\n\u003cp\u003eMethods for activating signal 2 are increasingly employed to enhance the effectiveness of immunotherapy.\u0026nbsp;Compared with PSMAxCD3 alone, 4-1BB co-stimulation, combined with PSMAxCD3, enhances CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, prolongs T-cell activation, and increases proliferation in larger tumors.\u003csup\u003e13\u003c/sup\u003e A class of BsAbs that mimics signal 2 by bridging the tumor antigen to the co-stimulatory CD28 receptor on T cells can substantially improve the effect of CD3 BsAbs.\u003csup\u003e14\u003c/sup\u003e CD80, a CD28 ligand, plays a crucial role in T-cell activation. Physiologically, CD80 interacts with PD-L1 in cis on primary activated dendritic cells, interfering with PD-L1/PD-1 binding and subsequently abrogating PD-1 function in T cells.\u003csup\u003e15\u003c/sup\u003e Certain tumor cells lack or under-express CD80 molecules on their surface, and upregulating CD80 expression in these tumor cells by drug action promotes antibody-mediated T-cell killing.\u003csup\u003e16\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eBased on the current evidence, we considered adding a second signal based on CD3 BsAbs. We incorporated the CD80 function into BsAbs because CD80 activates CD28 and binds to PD-L1. Therefore, we constructed a fusion protein containing scFvs targeting PSMA and CD3, together with the extracellular segment of CD80. This antibody exerted a favorable therapeutic effect on prostate tumors in a preclinical experimental study.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2-1 Construction of expression vectors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scFv-targeting PSMA was retrieved from a large yeast-displayed human single-chain antibody library.\u003csup\u003e17\u003c/sup\u003e ScFv-targeting CD3 and CD80 extracellular segment sequences were screened and obtained from the NCBI public database.\u003csup\u003e18\u003c/sup\u003e All fragment sequences were codon-optimized (Kingsley Bioscience and Technology Co. Ltd, China) for efficient expression in HEK293F cells. The scFv (PSMA) was ligated to the scFv (CD3) via a flexible linker 3 (G\u003csub\u003e4\u003c/sub\u003eS) using overlap PCR. The Fc segment of IgG1 was point-mutated (N297A) using the same technique.\u003csup\u003e19\u003c/sup\u003e Expression sequences were constructed and inserted into the mammalian expression vector pCMV3-C-Myc (Sino Biological, China). The full sequence information of antibodies was listed in Table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-2 Antibody expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plasmid was transiently transfected into HEK293F cells using FreeStyle\u0026trade; MAX Reagent (Thermo, USA) according to the kit instructions. The cells were cultured in shaker suspension (8% CO\u003csub\u003e2\u003c/sub\u003e, 37℃, 125rpm) using FreeStyle\u0026trade; 293 Expression Medium (Gibco, USA). After 7 days, the cell culture medium was collected and centrifuged at 3500rpm for 10 min. The supernatant was mixed with an equal amount of phosphate-buffered saline (PBS) and filtered using a 0.45-\u0026mu;m membrane (Millipore, USA). The filtrate was purified using a Protein A column (Beyotime, China) with an \u0026Auml;KTA protein purifier (GE, USA) to obtain the target antibody. The control antibody without the Fc segment structure was purified using a nickel ion chromatography column (Beyotime, China). The antibody concentration was determined using a NanoDrop2000 (Thermo Fisher Scientific, USA) microspectrophotometer after desalting and concentration; the antibody was frozen at \u0026minus;80\u0026deg;C after dispensing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePurified antibodies were separated by 12% SDS-PAGE under reducing conditions, followed by Coomassie Brilliant Blue staining of the gels and detection of the signals using a Chemiluminescent Imaging System (Bio-Rad ChemiDoc XRS+, USA). Under the same electrophoresis conditions, proteins were transferred to PVDF membranes for WB. After blocking with 5% BSA, membranes were incubated overnight at 4\u0026deg;C with anti-His antibody (Proteintech, China) or anti-human Fc antibody (Thermo Fisher Scientific, USA), followed by incubation with anti-mouse secondary antibody/HRP conjugate (Thermo Fisher Scientific, USA) for 2 hours at room temperature. Finally, the membranes were treated with an ultrasensitive luminescent agent (Solarbio, China). Signals were detected and analyzed by Image Lab software.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2-4 Cells and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman prostate cancer cell lines C4-2, 22Rv1, and PC-3(PSMA\u003csup\u003e-\u003c/sup\u003e)/(PSMA\u003csup\u003e+\u003c/sup\u003e) were cultured in a RPMI-1640 (Servicebio, China) medium with 10% fetal bovine serum (FBS) (Gibco, USA). Jurkat cells and human peripheral blood mononuclear cells (PBMCs) were cultured in a RPMI-1640 medium with 15% FBS. All cells were cultured at 37\u0026deg;C and under 5% CO\u003csub\u003e2\u003c/sub\u003e. PC-3(PSMA\u003csup\u003e+\u003c/sup\u003e) and 22Rv1-L were cell lines overexpressing the target gene (PSMA/Luciferase). With the approval of the Medical Ethics Committee of Xijing Hospital in Xi\u0026apos;an (KY20203128-1), peripheral blood samples were collected and PBMCs were isolated using Ficoll density gradient centrifugation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2-5 Detecting antibody binding activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC4-2/22Rv1/PC-3(PSMA\u003csup\u003e+\u003c/sup\u003e/PSMA\u003csup\u003e-\u003c/sup\u003e) cells were washed twice with PBS at 4\u0026deg;C, and the cell concentration was adjusted to 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/200 \u0026micro;L. Antibodies of each type were added at 10 nmol/L, and cells were co-incubated on ice for 30 min. After two PBS washes and incubation with anti-IgG-Fc fluorescent antibody (Abcam, UK) for 30 min in the dark, followed by one PBS wash, the binding activity of each antibody to the PSMA was determined by detecting the fluorescence intensity using a flow cytometer (Agilent NovoCyte, China). The binding activities to the CD3 antigen in Jurkat and human T cells were determined using the same method.\u003c/p\u003e\n\u003cp\u003eTriTE-N13 was incubated with C4-2/Jurkat cells at a final concentration of 0.001\u0026ndash;10 nmol/L (doubling dilution), and the average fluorescence intensity of C4-2/Jurkat cells was detected using a flow cytometry. The equilibrium dissociation constants (KD values) of TriTE-N13 were calculated using GraphPad Prism V8.4.3. Table S2 provides detailed information regarding the commercial antibodies used.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2-6 Determining lymphocyte activation by antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo detect antibody-mediated T-cell activation in peripheral blood mononuclear cells (PBMCs) without tumor cells, PBMCs were cultured in 48-well plates (Thermo Fisher Scientific, USA). Different concentrations of the target antibody were added to each experimental group. At 48/60/72 h, an anti-CD25/CD69/CD3 (Thermo Fisher Scientific, USA) fluorescent antibody was used for an incubation step, and the T-cell activation level was then detected using flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2-7 \u003cem\u003eIn vitro\u003c/em\u003e detection of antibody-mediated killing activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC4-2/22Rv1/PC-3(PSMA\u003csup\u003e+\u003c/sup\u003e/PSMA\u003csup\u003e-\u003c/sup\u003e) cells were selected and added to 48-well plates at 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well. Freshly extracted PBMCs were added according to the target efficiency ratio of E:T=4:1 (T is the initial cell number at the time of model establishment) after 24 h of incubation. Different concentrations (0.028/0.28/1.4/2.8/7/ 14 nmol/L) of the target antibody were added to each experimental grouping (NC/PCFC/CDFC/N2/N13) and incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture system was processed at 48/60/72 h according to the kit instructions (Servicebio Human IFN-gamma/IL-2/IL-6 ELISA Kit, China) to perform enzyme-linked immunosorbent assays (ELISA) of corresponding cytokines. The tumor cells were subjected to calcein/propidium iodide (PI) staining (Beyotime C2015M, China); the cell status was examined and photographed using a fluorescence microscope (Olympus FV3000, Japan). ImageJ software (V0.5.7) was used to analyze the results.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2-8\u003c/strong\u003e \u003cstrong\u003eDetermining antibody function in a three-dimensional (3D) tumor cell sphere model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC4-2/22Rv1 cells were added to the prepared 96-well plates at 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well in 150 \u0026mu;L of Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) (Servicebio, China) with 10% FBS, centrifuged at 1500 \u003cem\u003e\u0026times;g\u003c/em\u003e for 10 min, and cultured under routine conditions; the solution was changed after 3 days, and treatment experiments were initiated after 5\u0026ndash;7 days.\u003c/p\u003e\n\u003cp\u003eFreshly extracted PBMCs were resuspended in DMEM with 10% FBS and added to the well plate at the ratio of E:T=6:1. The corresponding antibody (7 nmol/L) was added according to the treatment group (NC/PBMC/PBMC+N2/PBMC+N13) and incubated at 24-h intervals using the EVOS M5000 live cell imager system (Thermo Fisher Scientific, USA) to observe the status of the tumor spheres and capture images (4\u0026times;). After 3 days, the supernatant in culture wells was collected and aspirated to detect the cytokine levels using an ELISA kit.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2-9 Establishing in vivo treatment of the transplantation tumor model in immunoreconstructed mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHumanized immune reconstitution was performed using severe combined immunodeficient mice (NOD/ShiLtJPrkdc\u003csup\u003eem26Cd52\u003c/sup\u003eIl2rg\u003csup\u003eem26Cd22\u003c/sup\u003e/Gpt; NCG).\u0026nbsp;Male\u0026nbsp;NCG mice\u0026nbsp;(4-5 weeks old)\u0026nbsp;were purchased from the\u0026nbsp;GemPharmatech Co., Ltd (Jiangsu, China). All mice weighed 20 \u0026plusmn; 2 g and were housed in the SPF barrier facility of the Fourth Military Medical University (Xi\u0026rsquo;an, China). All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of FMMU (Protocol No. 20220705) and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (2011).\u003c/p\u003e\n\u003cp\u003eHuman PBMCs (2\u0026times;10\u003csup\u003e7\u003c/sup\u003e) were transplanted into immuno-reconstructed NCG mice via the tail vein. The construction was deemed successful when the proportion of hCD45\u003csup\u003e+\u003c/sup\u003e T cells in mouse blood exceeded 25% (Figure S2). PBS-resuspended 22Rv1-L and (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e) tumor cells were transplanted by subcutaneous injection on the right side of the mice; tumor volume was calculated as [length\u0026times;width2]/2. Treatment experiments were initiated on day 7 after transplantation.\u003c/p\u003e\n\u003cp\u003eThe mouse models were randomly divided into four groups (NC/PBMC/N2/N13), and PBS/BiTE-N2/TriTE-N13 (bacterial filtration treatment, dose: 1 mg/kg) was injected through the tail vein once every 3 days. Tumor volume and mouse body weight were recorded every 3 days; the experiments were terminated when the tumor volume of the control group exceeded 2000 mm\u003csup\u003e3\u003c/sup\u003e. Mice were anesthetized using isoflurane mixed with oxygen, and the tumor was removed by blunt dissection through a 6\u0026ndash;8-mm longitudinal incision. After suturing the wound, the mice were euthanized under anesthesia. The tumor tissues were formalin-fixed and paraffin-embedded, sectioned, and stained using a fluorescent antibody against human CD8/CD45. The sections were scanned using an inverted phase-contrast fluorescence microscope (OLYMPUS, IX71, Japan) at a magnification of 20\u0026times;. The images were processed using Olympus FV31S-SW (V2.4.1.198). The culture medium was collected to determine the changes in human-derived cytokine levels in the serum using an ELISA kit. The heart, liver, kidney, lungs, and spleen were collected, fixed, sectioned, and stained with hematoxylin and eosin (HE); any damage to the organs and tissues was assessed.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2-10 Statistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as means \u0026plusmn; standard deviation (SD). Statistically significant differences were determined using specific tests in GraphPad Prism V8.4.3. Statistical analysis was performed using two-way analysis of variance (ANOVA) with a post hoc Tukey\u0026rsquo;s test for multiple comparisons. P\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3-1 Generating and characterizing the tri-specific antibody TriTE-N13\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe initially analyzed CD80 expression in various tumors using The Cancer Genome Atlas (TCGA) data.\u003csup\u003e20\u003c/sup\u003e CD80 expression was markedly higher in immunotherapy-sensitive tumors, such as kidney cancer, than in the adjacent tissues (Figure 1b). However, in prostate cancer, CD80 expression was not increased in tumor tissues when compared with that in adjacent tissues (Figure 1c). Furthermore, CD80 expression remained the most unaltered when compared with other highly expressed signaling pathway molecules involved in T cell activation in prostate cancer (Figure 1d). Collectively, these data indicated that signal 2 involving CD80 cannot fully activate T cells in prostate cancer.\u003c/p\u003e\n\u003cp\u003eAccordingly, we designed a bispecific antibody comprising four parts: two connected scFvs targeting CD3 and PSMA, an Fc segment with the BiTE-N297A mutation, and an extracellular group of CD80 fused to the C terminal of Fc (Figure 1e). To verify the effectiveness of this bispecific antibody, we designed control antibodies with different structures (Figure S1), including N1: PSMA(scFv)-CD3(scFv), BiTE-N2: PSMA-CD3-Fc (Figure 1f), PSMA(scFv)-Fc, and CD3(scFv)-Fc. The constructs were expressed and purified in 293F cells. TriTE-N13 is a fully human fusion antibody that effectively avoids anti-drug antibody (ADA) production during treatment. The molecular weight of TriTE-N13 is ~120 kDa (Figure 1g, h). Mass spectrometry analysis revealed that the target protein contained all the peptides from various functional regions, revealing that the antibody structure was identical and complete as designed (Table S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3-2 TriTE-N13 exhibits excellent binding activity on target cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree prostate cancer cell lines (C4-2/PC-3(PSMA\u003csup\u003e-\u003c/sup\u003e)/PC-3(PSMA\u003csup\u003e+\u003c/sup\u003e)) were used to detect the binding activity of antibodies to PSMA antigens. Jurkat and T cells sorted from human PBMCs were used to detect the CD3 binding activity. TriTE-N13 bound to PSMA and CD3 with high affinity (Figure 2a, b), and the binding activity of TriTE-N13 was comparable to that of PSMA-Fc and CD3-Fc. The binding activity of TriTE-N13 was comparable to that of BiTE-N2, indicating that the CD80 segment did not influence the binding activity of TriTE-N13 (Figure 2c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3-3 TriTE-N13 effectively activates T cells \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TriTE-N13-mediated activation effect was tested on T cells treated with TriTE-N13 or BiTE-N2 (Method S4). T cells treated with the TriTE-N13 antibody alone exhibited more pronounced proliferation after 4 days of culture, with only minimal proliferation observed upon treatment with BiTE-N2, the control antibody (Figure S3). CD25/CD69 expression on CD3\u003csup\u003e+\u003c/sup\u003e T cells was used as an indicator of T-cell activation. Treatment with TriTE-N13 activated ~20% of CD3\u003csup\u003e+\u003c/sup\u003e T cells. These results indicated that anti-CD3 and CD80 antibody components activate T cells in the absence of tumor cells (Figure 2d, e). We subsequently determined whether stronger activation was achieved when TriTE-N13 was added to the co-culture of T cells and PSMA-positive tumor cells. TriTE-N13 increased the T-cell activation intensity by 60% in a dose-dependent manner in the presence of PSMA-positive tumor cells (Figure 2f, g, i, j). However, the TriTE-N13-mediated activation effect on T cells was weak in the co-culture of PSMA-negative PC3 and T cells, indicating that T-cell activation by TriTE-N13 was antigen-specific (Figure 2h, k).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3-4 TriTE-N13 induces T-cell killing of PSMA\u003csup\u003e+\u003c/sup\u003e tumor cells \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we examined whether TriTE-N13 mediates the killing effect of T cells and used PI and calcein to indicate dead and live cells, respectively (Figure 3a). Compared with PSMA-negative PC-3 cells, PSMA-positive cells had a higher proportion of apoptotic cells after 60 h. TriTE-N13 and BiTE-N2 mediated a strong killing effect on all PSMA\u003csup\u003e+\u003c/sup\u003e tumor cells, which was gradually enhanced with the extension of co-culture time. Both the flow cytometry and CCK8 (Method S5) assay results revealed that TriTE-N13 exerted a more robust killing effect than BiTE-N2, with an average maximum killing rate of 80%. No significant cytotoxicity was observed in PSMA-negative PC-3 cells under the same interventions (Figure 3b, c).\u003c/p\u003e\n\u003cp\u003eWe further detected the interleukin (IL)-2, IL-6, and interferon (IFN)-\u0026gamma; levels in the culture supernatant and found that levels of all three cytokines were elevated in the TriTE-N13 and BiTE-N2 groups. TriTE-N13 effectively induced the release of these cytokines at low concentrations (Figure 3d). TriTE-N13 stimulated T-cell activation faster than BiTE-N2, as evidenced by the early detection of CD107a and TIM3 expression levels on CD8\u003csup\u003e+\u003c/sup\u003e T cells (Figure 3e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3-5 TriTE-N13 induces significant T-cytotoxic effects in the PSMA\u003csup\u003e+\u003c/sup\u003e tumor cell sphere model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough cell co-culture experiments at the two-dimensional (2D) level \u003cem\u003ein vitro\u003c/em\u003e demonstrated that TriTE-N13 effectively mediated the killing effect of T cells on tumor cell lines, the pavement-like arrangement of tumor cell lines did not mimic the 3D environment of solid tumors. We established a 3D model of globular aggregated tumor cell clusters and examined the ability of TriTE-N13 to induce T cell-mediated killing activity to restore the therapeutic environment in which the spatial structure of tumors impedes T cells from generating killing effects. After the C4-2/22Rv1 cell line was successfully selected to establish the tumor cell spherical aggregation cluster model (on day 4), sufficient amounts of antibody and fresh human PBMCs were added according to the grouping for co-culture. Changes in the cell sphere morphology were analyzed to determine the antitumor effects under different treatment conditions. TriTE-N13 induced a more substantial human PBMC killing effect on tumor cell spheres than BiTE-N2. Tumor cell spheres in the TriTE-N13 group were smaller than those in the BiTE-N2 group (Figure 4a, b). The culture supernatant of the TriTE-N13 group had higher levels of various cytokines at 48 h than the culture supernatants of other control groups (Figure 4c), indicating that TriTE-N13 was more effective than BiTE-N2 in tumor models with a 3D structure. However, the BiTE-N2-mediated tumor-killing effect in the 3D model was less than that in the conventional 2D model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3-6 TriTE-N13 effectively inhibits prostate tumor growth in a transplanted tumor model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e assays were performed to determine the antitumor function of the target antibody by establishing a xenograft tumor model in NCG mice with a reconstructed humanized immune system. At an initial treatment volume of \u0026lt;50 mm\u003csup\u003e3\u003c/sup\u003e, BiTE-N2 and TriTE-N13 significantly suppressed tumor growth (Figure 5a, b). Moreover, the tumor-suppressive effect of TriTE-N13 was superior to that of BiTE-N2 when the initial treatment volume of transplanted tumors was large (volume \u0026asymp;100 mm\u003csup\u003e3\u003c/sup\u003e). Upon discontinuation, the tumors in the BiTE-N2 group exhibited accelerated growth, whereas those in the TriTE-N13 group demonstrated sustained and effective suppression of tumor growth. This contrast further underscores the advantage of TriTE-N13 in maintaining therapeutic efficacy. (Figure 5c-f). The fluorescent staining (CD8/CD45) of tumor specimens and flow cytometry analysis of tumor tissue cell suspensions revealed that both the TriTE-N13 and BiTE-N2 groups exhibited a similar yet significant increase in lymphocyte infiltration when compared with the PBMC group (Figure 5g, h). However, the TriTE-N13 group showed the marked presence of infiltrating effector T-cells with elevated PD-1 expression, demonstrating the superior ability of TriTE-N13 to induce more effective T-cell activation (Figure 5i). To further validate the superiority of TriTE-N13 over BiTE-N2 in the large tumor volume model, we increased the initial treatment volume (~150 mm\u003csup\u003e3\u003c/sup\u003e), revealing that TriTE-N13 retained the more superior tumor suppression effect when compared with BiTE-N2 (Figure 5k-m).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3-7 TriTE-N13 did not cause excessive systemic toxic reactions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe focused on whether the target antibody exerted an overactivating effect on T cells \u003cem\u003ein vivo\u003c/em\u003e, given that TriTE-N13 comprised CD80 components that activate the second pathway, and \u003cem\u003ein vitro\u003c/em\u003e experiments revealed that TriTE-N13 activated T cells in human PBMCs. Weight changes in mice were recorded during treatment, revealing no significant inter-group differences (weight loss in the non-intervention group at the later stage may be related to the excessive tumor load) (Figure S4). Examination of hematoxylin-eosin stained sections of key organs at the end of the experiment found no significant differences in liver and kidney tissue damage among examined groups (Figure S5). ELISA was performed to detect serum levels of IL6/IFN-r in mice 21 days after initiating treatment, detecting no significant difference among examined groups. (Figure 5j).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe excellent performance of TCEs in hematological oncology has not been observed in solid tumors; this is mainly attributed to the complex microenvironment surrounding solid tumors and the \u0026ldquo;off-target effect,\u0026rdquo; which cannot be well resolved by TCEs in solid tissues. The prevailing strategy involves combining different immunotherapies with TCEs to achieve T-cell retargeting while increasing T-cell activation levels.\u003csup\u003e21\u003c/sup\u003e Although a combination of different therapies can elicit better efficacy than TCEs alone, the use of more drugs increases the possibility of off-target effects.\u003csup\u003e22\u003c/sup\u003e Therefore, we designed and produced a tri-specific TCEs containing a functional group of CD80 (N13) that adds CD80 to therapeutic TCEs with a PSMA-CD3 structure, aiming to increase the CD80 exposure in synapses when TCEs induces artificial synapse formation and enhance effector T-cell activation by TCEs via the CD28\u0026ndash;CD80 pathway, thereby improving the efficacy of immunotherapy on prostate tumors.\u003c/p\u003e\n\u003cp\u003eTriTE-N13 exhibited excellent binding activity to both PSMA\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003e cells and effectively activated T cells in PBMCs to produce a dose-dependent killing effect on PSMA\u003csup\u003e+\u003c/sup\u003e prostate tumor cells \u003cem\u003ein vitro\u003c/em\u003e. We observed similar cytotoxic effects in an \u003cem\u003ein vitro\u003c/em\u003e tumor cell sphere model. Using a xenograft tumor model of immuno-reconstructed mice, we showed that TriTE-N13 could effectively suppress tumor growth. Furthermore, TriTE-N13 and BiTE-N2 increased T-cell infiltration in the tumor, although PD-1 expression on intratumoral T cells was elevated in the TriTE-N13 group when compared with that in other groups, suggesting that TriTE-N13 could more efficiently activate immune cell infiltration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe suppressive tumor microenvironment of solid tumors requires that immunotherapy fully activates the immune system.\u003csup\u003e23\u003c/sup\u003e However, it is important to reconcile enhancing the immune activation level to increase efficacy while reducing the activation level to decrease side effects. TriTE-N13 is designed to reinforce the T-cell activation level by increasing CD80 exposure at the immune synapse. In our \u003cem\u003ein vitro\u003c/em\u003e co-culture experiments, TriTE-N13 elicited T-cell activation at a lower concentration level than BiTE-N2; TriTE-N13 mediated higher levels of cytokine release in the tumor cell sphere model, with more pronounced tumor sphere suppression. Although TriTE-N13 activated T cells prior to immune synapse formation, the activated T cells possess PSMA targeting properties because of surface enrichment with TriTE-N13 (carrying the PSMA antigenic recognition site). \u003cem\u003eIn vivo\u003c/em\u003e, TriTE-N13 did not induce severe side effects when compared with BiTE-N2 while exerting a potent tumor-suppressive effect. The next stage of our research will focus on determining whether CD80 in TriTE-N13 mediates CD28 co-stimulatory signaling while further regulating the immune response via the PD-1/CTLA-4 pathway.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e#Y. Jiang, H. Li contributed equally to this work. This study was supported by the National Natural Science Foundation of China (No. 82220108004; 82173204; 82203633), the Innovation Capability Support Program of Shaanxi (2023-CX-TD-72; 2021TD-39; 2020PT-021), and the Natural Science Basic Research Program of Shaanxi (2022JZ-62).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, et al. 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T cell-engaging therapies - BiTEs and beyond.\u003cem\u003e Nat Rev Clin Oncol. \u003c/em\u003e2020 Jul;17(7):418-434. \u003c/li\u003e\n\u003cli\u003eTanaka A, Sakaguchi S. Regulatory T cells in cancer immunotherapy. \u003cem\u003eCell Res.\u003c/em\u003e 2017 Jan;27(1):109-118.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"T cell engager, CD80, prostate cancer, tri-specific antibodies, PSMA/CD3 antigens","lastPublishedDoi":"10.21203/rs.3.rs-5391004/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5391004/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eT-cell engagers (TCEs) are the most examined bispecific antibodies that recognize and target effector T cells to generate immune synapses against tumor cells, thereby resulting in T-cell activation and tumor killing. However, the inhibitory influence of the tumor microenvironment on TCEs' functionality has impeded their utilization in solid tumor therapies. To circumvent this limitation, we enhanced bispecific prostate-specific membrane antigen (PSMA)/CD3 TCEs by incorporating the extracellular segment of CD80 to overcome T-cell inhibition in prostate cancer by delivering a second activation signal to T cells, designating this trifunctional antibody as TriTE-N13.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods：\u003c/strong\u003eUsing gene editing and eukaryotic expression techniques, we developed the fully humanized fusion antibody TriTE-N13. \u003cem\u003eIn vitro\u003c/em\u003e, we assessed its effects on T-cell activation, proliferation, and cytotoxicity, and tested the T-cell targeting cytotoxicity function of TriTE-N13 against PSMA\u003csup\u003e+\u003c/sup\u003e tumor cells in both cell co-culture models and tumor cell spheroid models. Furthermore, in humanized immune-reconstituted mouse models, we evaluated the \u003cem\u003ein vivo \u003c/em\u003eefficacy and safety of TriTE-N13 against prostate cancer xenografts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e \u003cem\u003eIn vitro\u003c/em\u003e, TriTE-N13 can notably activate human T cells and exhibits excellent binding activity to human PSMA and CD3 antigens. Meanwhile, TriTE-N13 can mediate T-cell-induced cytotoxicity against PSMA-positive prostate cancer cells. \u003cem\u003eIn vivo\u003c/em\u003e, we demonstrated that TriTE-N13 significantly reduced tumor volume compared to bispecific TCEs when treating initially large-sized tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur data suggest that the incorporation of CD80 as a second pathway activator significantly enhances the solid tumor-killing effect of TCEs, thereby positioning TriTE-N13 as a promising immunotherapy candidate for the treatment of advanced prostate cancer.\u003c/p\u003e","manuscriptTitle":"A multifunctional T-cell engager containing CD80 enhances prostate cancer treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-18 14:45:20","doi":"10.21203/rs.3.rs-5391004/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-10T16:12:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-09T21:46:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221779701414386513471909019167967686105","date":"2025-04-05T18:00:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-03T22:24:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-03T07:57:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47041501836765680259214121974930853048","date":"2025-04-02T23:22:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153569339302305759482638830092334773671","date":"2025-04-01T11:34:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77550539175979311328100014761296565278","date":"2025-03-31T18:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166199042673510415786636411264388715506","date":"2025-03-31T17:47:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99601364607736634961362750842150099398","date":"2025-01-10T16:38:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154809692095285050058056242696618888142","date":"2024-11-17T18:36:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-15T18:11:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-05T06:30:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-05T06:29:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2024-11-04T23:36:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4d8b9ea1-3517-41bb-bdab-8ba455061938","owner":[],"postedDate":"November 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T16:00:58+00:00","versionOfRecord":{"articleIdentity":"rs-5391004","link":"https://doi.org/10.1007/s00262-025-04121-0","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-07-23 15:57:32","publishedOnDateReadable":"July 23rd, 2025"},"versionCreatedAt":"2024-11-18 14:45:20","video":"","vorDoi":"10.1007/s00262-025-04121-0","vorDoiUrl":"https://doi.org/10.1007/s00262-025-04121-0","workflowStages":[]},"version":"v1","identity":"rs-5391004","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5391004","identity":"rs-5391004","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}