Modular construction of bispecific antibodies through bioconjugation for T cell-based immunotherapy

Modular construction of bispecific antibodies through bioconjugation for T cell-based immunotherapy | 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 Modular construction of bispecific antibodies through bioconjugation for T cell-based immunotherapy Shou-qing Sun, Lian Wang, Jing Xia, Shun Li, Jian-min Zhu, Cai-Wen Duan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8371642/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bispecific antibodies (bsAbs) represent a groundbreaking advance in antibody engineering, overcoming therapeutic limitations of monoclonal antibodies through dual-targeting capabilities. However, their clinical translation is often hindered by structural and manufacturing complexities. In this study, we developed a modular bsAb platform utilizing the SpyCatcher-SpyTag site-specific coupling system for T cell-based immunotherapy. We successfully assembled functional bsAbs by conjugating tumor-targeting SpyCatcher-fused antibodies (anti-CD19, FMC63-Fc-SC or anti-HER2, ZHER-SC) with SpyTag-fused anti-CD3 domains (7G03-ST). These bsAbs effectively killed CD19-expressing leukemic cells and HER2-positive solid tumor cells. Furthermore, through substituting the anti-CD3 domain (e.g., OKT3-ST), we readily generated bsAbs targeting CD19, HER2, PD-L1 and EGFR, all of which specifically engaged their respective tumor antigens. More importantly, bsAbs constructed via this modular strategy significantly enhanced the cytotoxicity of CAR-T cells. In conclusion, this flexible bioconjugation platform offers a reliable and efficient technical solution for the rapid development of diverse bispecific antibodies. Bispecific antibodies SpyCatcher-SpyTag system T cell-based immunotherapy CAR-T cells Site-specific Conjugation. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Bispecific antibodies (bsAbs) have emerged as a highly promising class of therapeutic agents, introducing revolutionary changes to the fields of medicine and biotechnology 1 – 3 . The unique molecules, engineered to bind two distinct antigens simultaneously 3 – 5 , function by bridging tumor-associated antigens (TAAs) and immune effector cells such as T cells. BsAbs address the limitations of monospecific antibodies, enabling major histocompatibility complex-independent redirection of cytotoxicity 6 . Such a dual-targeting strategy has demonstrated significant clinical efficacy in hematological malignancies; for instance, blinatumomab (anti-CD19/CD3) achieves a 43% complete remission rate in relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) 7 . The modular nature of bsAbs further supports the construction of customized immune synapses, establishing their position as a core tool in next-generation immuno-oncology 2 , 8 , 9 . This dual-targeting feature opens up broad potential medical applications, particularly in the treatment of complex diseases such as cancer and autoimmune disorders 10 . However, the structural complexity and manufacturing of bispecific antibodies present significant challenges. The unnatural chain combinations frequently lead to mispairing, aggregation, and low expression yield, imposing stringent requirements on manufacturing processes and quality control 11 . Current development efforts are increasingly focusing on optimizing designs to enhance safety, improve pharmacokinetic properties, and expand therapeutic indications 12 . Through development of more stable humanized frameworks and implementation of modular assembly platforms, bispecific antibodies could assume a more central role in the current era of precision medicine, ultimately fulfilling their considerable therapeutic potential 3 , 12 . The combined application of CAR-T (Chimeric Antigen Receptor T cells) cells and bispecific antibodies is a promising strategy in cancer immunotherapy. CAR-T cells have demonstrated considerable efficacy in the treatment of certain hematological malignancies 13 , 14 ; however, their efficacy in solid tumors is still limited by antigen heterogeneity 15 , immunosuppressive microenvironments 16 , and on-target/off-tumor toxicity 17 . To enhance CAR-T function, a strategy integrating BsAbs has emerged 18 , 19 . BsAbs can recruit endogenous T cells to CAR-T-resistant tumor subsets or target alternative TAAs 20 , thereby expanding antigen coverage and reducing immune escape 21 . This further improves the specificity and efficacy of CAR-T cell-mediated killing 22 . Preclinical studies have shown that anti-CD20/CD3 BsAbs can enhance the efficacy of CD19 CAR-T cells in heterogeneous lymphoma models significantly 23 , 24 , highlighting the potential of the integrated treatment platform. Traditional BsAb production is confronted with scalability, stability, and homogeneity challenges. The SpyCatcher-SpyTag system is a powerful protein-ligation technology with numerous applications in protein engineering and biotechnology 25 – 27 . It is based on a modified domain from a Streptococcus pyogenes surface protein, SpyCatcher, which specifically recognizes a cognate 13 - amino - acid peptide, SpyTag 26 , 28 . Upon recognition, an irreversible covalent isopeptide bond forms between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag 26 , 27 , 29 . The reaction occurs rapidly and efficiently under physiological conditions, and is insensitive to numerous factors such as buffer composition, temperature, and pH 28 . The SpyCatcher-SpyTag system offers several advantages over traditional protein-conjugation methods 29 , 30 . Formation of a covalent bond ensures a stable and robust linkage between proteins, which is crucial for applications such as construction of multi-protein complexes or modification of proteins with functional moieties 29 . In addition, the small size of the SpyTag peptide (only 13 amino acids) minimizes potential interference with the structure and target protein function. Furthermore, the system is highly versatile and can be used to conjugate a wide range of proteins, both in vitro and in vivo 31 . The aim of the this study was to modularly construct various bispecific antibodies to effectively activate T cells or CAR-T cells and mediate their killing effect on tumor cells. The use of the SpyCatcher-SpyTag system allows for precise and efficient construction of the bispecific antibodies, which is expected to address some challenges associated with traditional bispecific antibody preparation methods. Bispecific antibody was used to conjugate an antibody fusion protein targeted tumor with SpyCatcher to an antibody fusion protein incorporating the anti-CD3 domain (7G03 and OKT3) and SpyTag. BsAbs targeting HER2, CD19, PDL1, and EGFR were generated by constructing ZHER (anti-HER2), FMC63 (anti-CD19), anti-PDL1, and anti-EGFR bispecific antibodies, respectively, and the bsAbs were applied to target tumor cells expressing CD19, PDL1, or EGFR. Furthermore, modular bioconjugated construction of bispecific antibodies was used to enhance CAR-T cell cytotoxicity. Our research could not only contribute to the development of novel immunotherapeutic strategies but also provide insights that could facilitate the application of the SpyCatcher-SpyTag system in antibody engineering. RESULTS Modular Construction and Cytotoxic Efficacy of the Bispecific Antibodies To target human T cells, an anti-CD3 nanobody fused with SpyTag, named 7G03-SpyTag (7G03-ST), was designed. To achieve efficient site-specific conjugation via the SpyCatcher-SpyTag system, an anti-CD19 scFv-Fc antibody fused with SpyCatcher was expressed and purified, and termed FMC63-Fc-SpyCatcher (FMC63-Fc-SC) (Fig. 1 A). Since SpyCatcher and SpyTag spontaneously recognize and form a stable covalent bond under conventional conditions, the SpyTag-modified 7G03 could be site-specifically conjugated with FMC63-Fc-SC upon mixing, yielding the modular bispecific antibody FMC63-7G03 (Fig. 1 B, C). To obtain and cultivate T cells, T cells were isolated from healthy donors and stimulated in vitro with CD3/CD28 antibodies (5 µg/mL) for 48 h. To evaluate the T cell-mediated cytotoxicity induced by FMC63-7G03, the activated T cells were then co-cultured with Nalm6 cells for 24 h with bsAbs. Cytotoxicity against Nalm6 cells was assessed by flow cytometry. The results demonstrated that FMC63-7G03 effectively redirected activated T cells to kill Nalm6 cells (Fig. 1 D). In contrast, FMC63-7G03 alone did not affect the Nalm6 cells viability (Fig. 1 E). Furthermore, the expression of T cell activation markers (CD25 and CD69) was examined following co-culture with Nalm6 cells in the presence or absence of specific antibodies. Significant upregulation of CD25 and CD69 was observed only when FMC63-7G03 was present (Fig. 1 F, FigS.1A). Additionally, after 24 h of co-culture with tumor cells in the presence of FMC63-7G03, ELISA analysis revealed a marked increase in the secretion of effector cytokines—including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2—in the culture supernatant (Fig. 1 G). The FMC63-7G03 bispecific antibody, successfully constructed using the nanobody 7G03-ST, can direct T cells to eliminate CD19-expressing Leukemia cells. The findings indicate that modularly constructed bispecific antibodies could effectively activate T cells and direct them to specifically eliminate antigen-expressing tumor cells. Modular Constructed Bispecific Antibodies Mediates T Cell Killing of Solid Tumor Cells To investigate whether the modular approach could be extended to other targets, the anti-HER2 ZHER-SpyCatcher (ZHER-SC) protein, an affibody antibody (Fig. 2 A), and this was site-specifically conjugated with 7G03-ST to form the modular bispecific antibody ZHER-7G03 (Fig. 2 B, C). To assess whether ZHER-7G03 could redirect T cells to kill antigen-matched tumor cells, activated T cells were co-cultured with Daoy (HER2 + ) target cells at an effector-to-target (E:T) ratio of 1:1 in the presence of 1000 ng/mL ZHER-7G03 for 24 h. Target cell death was quantified by flow cytometry, revealing specific lysis of approximately 60% of the cells in the ZHER-7G03 group, which was significantly higher than those in the control groups (Fig. 2 D). Incubation with ZHER-7G03 alone did not affect tumor cell viability (Fig. 2 E). Furthermore, T cells treated with ZHER-7G03 exhibited significantly elevated surface expression of the activation markers CD25 and CD69 (Fig. 2 F, FigS. 2 A). ELISA analysis of culture supernatants also demonstrated markedly increased levels of the effector cytokines TNF-α, IFN-γ, and IL-2 (Fig. 2 G). The results indicate that ZHER-7G03, constructed via the SpyCatcher-SpyTag system, is biologically active. It not only induces antigen-specific T cell activation but also promotes secretion of cytotoxic effector molecules, enabling precise elimination of HER2 + solid tumor cells. Validation of Versatility and Tumor Killing Efficacy of the Modular Bispecific Antibody Construction Platform To validate the versatility of the modular bispecific antibody architecture presented, 7G03-ST was replaced with OKT3-Fc-SpyTag (OKT3-Fc-ST), an scFv-Fc fusion antibody that also targets CD3 to improve stability by Fc segments. OKT3-Fc-ST was efficiently and stably conjugated with the scFv antibody FMC63-Fc-SC and the affibody ZHER-SC to generate FMC63-OKT3 (Fig. 3 A, Figs. S3A) and ZHER-OKT3 (Fig. 3 F, Figs. S3E). Pre-stimulated T cells were co-cultured with target cells at a 1:1 ratio for 24 h, and the results indicated that OKT3-Fc-ST alone could activate T cells moderately; however, in the presence of FMC63-OKT3 or ZHER-OKT3, T cell-mediated cytotoxicity was enhanced significantly (Fig. 3 B, G, Figs. S3B, F). Neither bispecific antibody affected tumor cell viability alone (Figs. S3C, G). Tumor cell killing exhibited a dose-dependent response to concentration of bispecific antibodies in the system (Fig. 3 C, H). Moreover, T cells treated with the bispecific antibodies exhibited significantly elevated expression of the activation markers CD25 and CD69 (Fig. 3 D, I, Figs. S3D, H). ELISA analysis of culture supernatants revealed markedly increased levels of effector cytokines—TNF-α, IFN-γ, and IL-2 (Fig. 3 E, J). Furthermore, anti-EGFR and anti-PDL1 nanobodies fused with SpyCatcher: aEGFR-SC and aPDL1-SC were constructed. Both nanobodies bound specifically to Daoy cells expressing their respective targets (Fig. 4 A). They were conjugated with OKT3-Fc-ST to form aEGFR-OKT3 and aPDL1-OKT3 (Fig. 4 B, Figs. S4A, B). Neither bispecific antibody influenced tumor cell growth alone (Figs. S4E, F), yet both effectively redirected T cells to kill target cells in a concentration-dependent manner (Fig. 4 C, D, Figs. S4C, D). Additionally, they promoted T cell activation and effector cytokine secretion (Fig. 4 E–H, Figs. S4G). The results confirm the broad versatility of the modular bispecific antibody construction platform based on the SpyCatcher-SpyTag system. By modular assembly of CD3-targeting OKT3-Fc-ST with various targeting moieties—including FMC63, ZHER, aEGFR, and aPDL1—multiple bispecific antibodies were constructed successfully, each effectively mediating T cell killing of antigen-matched target cells. The platform provides a reliable and flexible strategy for rapid generation of diverse bispecific antibodies. Modular Construction of Bispecific Antibodies through Bioconjugation Enhances CAR-T Cell-Mediated Cytotoxicity To determine whether modular bispecific antibodies can enhance CAR-T cell cytotoxicity, second-generation CD19-specific CAR-T cells incorporating 4-1BB as a costimulatory domain were generated. The CD19 CAR construct consisted of an extracellular anti-CD19 (FMC63) single-chain variable fragment (scFv), a transmembrane domain, and intracellular signaling domains derived from human 4-1BB and CD3ζ (Fig. 5 A). After 48-h stimulation, T cells were transduced with a CD19 CAR lentiviral vector, resulting in approximately 60% CAR-positive cells (Fig. 5 B). In addition, Nalm6 tumor cells were engineered to overexpress PDL1 (Nalm6-PDL1) (Fig. 5 C). When T cells or CAR-T cells were co-cultured with Nalm6-PDL1 for 24 h, aPDL1-OKT3 further activated CD8 + CAR-T cells and enhanced their tumor-killing capacity (Fig. 5 D, E). To further examine T cell activation dynamics, CAR-T cells were restimulated with CD3/CD28 antibodies for 48 h. This not only increased the proportion of CD8 + CAR-T cells and CAR positivity (Fig. 5 F) but also induced PD-1 expression (Fig. 5 G), indicating emergence of a pre-exhaustion phenotype in a subset of T cells, following strong activation. Subsequently, the CAR-T cells were co-cultured with Nalm6-PDL1 cells for 24 h. Although the cytotoxic efficacy of CAR-T cells was reduced partially due to the exhausted phenotype, the presence of aPDL1-OKT3 effectively restored T cell-mediated tumor killing (Fig. 5 H). In addition, second-generation B7H3-specific CAR-T cells were established, also containing the 4-1BB costimulatory domain (Fig. 5 I). Co-culture of T cells or CAR-T cells with Daoy cells for 24 h demonstrated that aEGFR-OKT3 similarly enhanced CAR-T cell activation and tumor cell killing (Fig. 5 J, K). The observations suggest that modular construction of bispecific antibodies through bioconjugation can partially overcome or compensate for functional impairments in early-exhausted CAR-T cells. CONCLUSIONS In the present study, an scfv fusion protein incorporating FMC63 (anti-CD19) and SpyCatcher domains was initially expressed, and the FMC63 domain specifically targets the CD19 antigen, whereas the SpyCatcher domain enables conjugation with the nanobody 7G03-ST, thereby directing T cells to recognize and eliminate CD19-positive tumor cells. Furthermore, additional bispecific antibodies were generated by expressing fusion proteins containing either an anti-HER2 affibody, an anti-PDL1 nanobody, or an anti-EGFR nanobody, which were site-specifically conjugated with 7G03-ST or OKT3-ST. In vitro cytotoxicity assays demonstrated that all the bispecific antibodies effectively mediated T cell-induced tumor cell killing. Notably, the bispecific antibody aPDL1-OKT3 not only enhanced CAR19-T cell-mediated cytotoxicity against Nalm6-PDL1 tumor cells but also restored the functional capacity of early-exhausted CAR-T cells partially. Collectively, the findings indicate that modular proteins constructed via the SpyCatcher/SpyTag system can effectively bridge T cells and tumor cells through a bispecific binding mode. Such interaction induces antigen-specific T cell activation and promotes secretion of cytotoxic effector molecules, leading to precise tumor cell elimination. The modular design strategy, leveraging efficient and stable site-specific conjugation, establishes a transferable technological paradigm for engineering bispecific antibodies. Its versatility demonstrates considerable potential in translational immunotherapy applications across diverse antigen targets. MATERIALS AND METHODS Reagents and Materials Phosphate buffered saline (PBS), Isopropyl β-d-1-thiogalactopyranoside(IPTG), Kanamycin B, imidazole, Coomassie brilliant blue, and Ni-IDA-Sefinose resin were purchased from Sangon (Shanghai, China). DAPI was obtained from Beyotime Biotechnology (Jiangsu, China). MaxFB, MaxFA6, and HEK293 MaxD media were purchased from MediumBank Biotechnology Co., Ltd. (Shanghai, China). Flow cytometry antibodies and dyes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Minimum Essential Medium (MEM), MEM Non-Essential Amino Acids Solution (NEAA), Dulbecco’s Modified Eagle Medium, RPMI 1640 basic Medium, and Fetal Bovine Serum (FBS) were purchased from Gibco (Frederick, MD, USA). X-VIVO 15 was purchased from Lonza (Basel, Switzerland). Serum-free CELLSAVING and penicillinstreptomycin (P/S) were obtained from NCM Biotech (Suzhou, China). Human CD3 (humanized OKT3) and CD28 antibodies were purchased from Sino Biological Inc. (Beijing, China). Cell lines and primary cells Cell lines: The human medulloblastoma cell line Daoy and the B-cell acute lymphoblastic leukemia cell line Nalm6 were obtained from the American Type Culture Collection (ATCC). Daoy cells were maintained in MEM medium supplemented with 10% FBS, 1% penicillin–streptomycin (P/S), and 1% non-essential amino acids (NEAA). Nalm6 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% P/S. The human embryonic kidney epithelial cell line HEK293T was acquired from the National Collection of Authenticated Cell Cultures and grown in HEK293 MaxD medium supplemented with 3% MaxFA6 and 0.3% MaxFB. All cell lines were incubated at 37°C in a humidified atmosphere with 5% CO₂. Peripheral Blood Mononuclear Cells and T Cell Isolation: Human primary T cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) obtained from healthy donors. Whole blood was processed by density gradient centrifugation using a human PBMC isolation buffer. The isolated PBMCs were cultured in X-VIVO 15 medium containing 4% FBS and 300 U/mL recombinant human IL‑2 (SL pharm, Beijing, China). T cell activation was induced by a 48-h stimulation with anti-human CD3 and anti-human CD28 antibodies, each at a concentration of 5 µg/mL. Following activation, T cells were maintained in culture for 5–12 d prior to subsequent experiments. Protein Production and Purification The OKT3-Fc-ST construct, comprising OKT3, Fc, SpyTag, and a His-Tag, was cloned into the protein expression vector pSB. Similarly, the FMC63-Fc-SC construct—consisting of FMC63, Fc, SpyCatcher, and a His-Tag—was also inserted into the pSB vector. The corresponding nucleotide sequences are provided in Table S1 . Transfection of the expression vectors into HEK-293T cells was performed using Lipofectamine 3000 transfection reagent (Life Technologies, Carlsbad, CA, USA), and stable cell lines were established under selection with 2 µg/mL puromycin. Additionally, 7G03-SpyTag, ZHER-SpyCatcher, aPDL1-SpyCatcher, and aEGFR-SpyCatcher were constructed as fusions of the respective antibody fragments with SpyTag or SpyCatcher and a His-Tag, and were cloned into the pET28A expression vector. The sequences of the constructs are also listed in Table S1 . The recombinant vectors were transformed into Escherichia coli for protein expression. Positive clones were cultured in Luria-Bertani liquid medium supplemented with 10 µg/mL kanamycin at 37°C until the OD600 reached 1.0. Protein expression was induced by adding 1 mM IPTG, followed by overnight incubation at 16°C. Bacterial cells were harvested by centrifugation at 4000 ×g for 5 min and lysed via ultrasonication. The soluble fraction was obtained by centrifugation at 12,000 ×g for 30 min at 4°C. Protein purification was performed using Ni-IDA gravity chromatography columns. Culture supernatants from HEK-293T cells or bacterial lysates were centrifuged at 4°C (4000 ×g, 5 min) and applied to the columns. After washing with 20 or 40 mM imidazole in PBS, the target proteins were eluted using 200 or 500 mM imidazole in PBS. Elution fractions were analyzed by SDS-PAGE with Coomassie blue staining, and those showing the expected protein bands were pooled. Proteins were concentrated and buffer-exchanged into PBS using an Amicon Ultra-4 10 kDa centrifugal filter device (Merck, Rahway, NJ, USA) with three dialysis steps. Purified proteins were aliquoted and stored at − 20°C for long-term storage or at 4°C for immediate use. Site-Specific Ligation of SpyCatcher‑Tagged Proteins with SpyTag‑Containing Constructs The tumor-targeting protein fused to SpyCatcher was mixed with either 7G03-SpyTag or OKT3-Fc-SpyTag at a 1:1 molar ratio (SpyTag:SpyCatcher). The reaction was carried out with gentle rotation at 37°C for 2 h, followed by further incubation overnight at 4°C under continuous gentle rotation. To confirm formation of the conjugate, the products were analyzed by SDS‑PAGE with Coomassie blue staining, and samples exhibiting the expected molecular weight bands were selected for subsequent use. In Vitro Cytotoxicity Study. Tumor cells were first labeled with 2.5 µM Cell Proliferation Dye (CPD) or 0.25 µM CFSE for 15 min at 37°C. Subsequently, T cells and labeled tumor cells were co-cultured in 48-well plates at a 1:1 effector-to-target (E:T) ratio. Phosphate-buffered saline (PBS), modular bispecific antibodies (bsAbs), or corresponding controls were then administered, and the plates were incubated at 37°C for 24 h. Following incubation, cells were stained with 5 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 4°C, washed once with PBS, and subjected to flow cytometric analysis. Tumor cell lysis was determined by quantifying the populations of CPD⁺/DAPI⁺ or CFSE⁺/DAPI⁺ cells. Generation of CAR-T cells HEK-293T cells were seeded in 100-mm cell culture dishes and cultured overnight. The cells were then co-transfected with CAR19 or CARB7H3 lentiviral vectors, along with the packaging plasmids psPAX2 and pMD2.G, using a liposome-based transfection reagent. Viral supernatant was collected at 48 and 72 h post-transfection and concentrated using polyethylene glycol 8000 (PEG8000). The resulting viral precipitate was resuspended in phosphate-buffered saline (PBS). For T cell transduction, primary human T cells were stimulated with anti-human CD3 and CD28 antibodies for 48 h, followed by exposure to the respective lentiviral particles in the presence of 10 µg/mL polybrene. Transduction efficiency was evaluated 48 h later by flow cytometry based on the percentage of CAR-positive cells. Statistical Analyses Two-tailed unpaired t-tests were used to compare two groups. One-way Analysis of Variance was used to compare three or more groups in a single condition. All values in the study were expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). Differences with P < 0.05 (*) P < 0.01(**) P < 0.001(***), and **** P < 0.0001(****) were considered significant Declarations ASSOCIATED CONTENT Supporting Information . Additional data, including the sequences of protein utilized in this work and additional experimental results. Author Contributions S.-Q.S, L.W., and J.X. designed and performed the experiments, interpreted the results, and prepared the manuscript. S.L. and J.-M.Z performed some of the experiments. K.-M.C. and C.-W.D. conceived of the study, designed experiments, supervised research, and wrote the manuscript. S.-Q.S, L.W., and J.X. contributed equally to this work. Funding This research was supported by the National Key R&D Program of China (NO. 2021YFA1100800 to A.-B.L.), the Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China (Grant No. 2023ZQNZD012) and the Natural Science Foundation of Fujian Province (No. 2024J011122). ACKNOWLEDGMENT We thank the Pediatric Translational Medicine Institute of Shanghai Children’s Medical Center for technical support. Ethical Approval Permission from the Institutional Animal Ethical Committee was received before making these experiments. Data Availability Statement All data generated or analyzed during this study are included in this published article and its supplementary information files. Consent to Participate Not applicable. Consent for Publication Not applicable. Conflict of Interest The authors declare no competing interests. References Holliger, P., & Hudson, P. J. (2005). Engineered antibody fragments and the rise of single domains. 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Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. P Natl Acad Sci USA , 109 (12), E690–E697. Li, L., Fierer, J. O., Rapoport, T. A., & Howarth, M. (2014). Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. Journal Of Molecular Biology , 426 (2), 309–317. Keeble, A. H., & Howarth, M. (2020). Power to the protein: enhancing and combining activities using the Spy toolbox. Chemical Science , 11 (28), 7281–7291. Scheme 1 Scheme 1 is available in the Supplementary Files section. Supplementary Files Supplementalinformation.docx image6.png Scheme. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-8371642","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":568410258,"identity":"31dfd4e4-25ef-45b6-942e-bb3fb2a1421e","order_by":0,"name":"Shou-qing Sun","email":"","orcid":"","institution":"Shanghai Children's Medical Center Affiliated to Shanghai Jiaotong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shou-qing","middleName":"","lastName":"Sun","suffix":""},{"id":568410259,"identity":"55766564-ef12-4211-a185-9998e9676bbe","order_by":1,"name":"Lian 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06:29:14","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101325,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/e52e65c407a08cf8ed04b8da.html"},{"id":99496354,"identity":"2727171b-704b-41f3-a68f-13df5f21a5b9","added_by":"auto","created_at":"2026-01-05 06:29:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and functional characterization of the modular bispecific antibody FMC63-7G03.\u003c/strong\u003e (A) Binding of FMC63-Fc-SC with CD19-positive Nalm6 cells was detected by flow cytometry. (B) Schematic illustration of the construction process of FMC63-7G03. (C) The bispecific antibody assembly was analyzed by SDS-PAGE with Coomassie blue staining. (D) Cytotoxicity assay of the Nalm6 incubated with PBS, FMC63-Fc-SC, 7G03-ST or FMC63-7G03 in the presence of T cells, the effector cells: target cells (E:T = 1:1). The antibody concentration is 1000 ng/ml. (E)Cytotoxicity against Nalm6 cells incubated with FMC63‑7G03 in the absence of T cells (1000 ng/mL). (F) Quantification of activation markers in T cells after incubation with Nalm6 cells, and plus PBS, FMC63-Fc-SC, 7G03-ST or FMC63-7G03 for 24 h. The antibody concentration is 1000 ng/ml. (G) T cells were co-cultured with Nalm6 cells for 24 hours in the presence or absence of FMC63-7G03, and cytokine levels in the culture supernatant were quantified by ELISA. All cytotoxicity assays were assessed by flow cytometry for DAPI staining, and all data are presented as means ± s.d., n = 3.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/4939791fffe4f31cbf2fcc14.png"},{"id":99496358,"identity":"2f54b6e5-476c-49cd-9fc6-77345fbedd3b","added_by":"auto","created_at":"2026-01-05 06:29:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZHER-7G03 mediates T cell killing of solid tumor cells\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Binding of ZHER‑SC to HER2‑positive Daoy cells and HER2‑negative Nalm6 cells was assessed by flow cytometry. (B) Schematic diagram illustrating the assembly strategy of ZHER‑7G03. (C) SDS‑PAGE with Coomassie blue staining was used to analyze the assembled bispecific antibody. (D) Cytotoxicity against HER2‑expressing Daoy cells incubated with PBS, ZHER‑SC, 7G03‑ST, or ZHER‑7G03 in the presence of T cells at an effector-to-target (E:T) ratio of 1:1. Antibody concentration was 1000 ng/mL. (E) Cytotoxicity against Daoy cells incubated with ZHER‑7G03 in the absence of T cells (1000 ng/mL). (F) Expression of T cell activation markers after 24 h co‑culture with Daoy cells in the presence of PBS, ZHER‑SC, 7G03‑ST, or ZHER‑7G03 (1000 ng/mL). (G) Cytokine levels in the supernatant of T cells and Daoy cells co‑cultured for 24 h with or without ZHER‑7G03, as quantified by ELISA. In all cytotoxicity assays, cell death was determined by DAPI staining and flow cytometry. Data are presented as mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/5bbff18c1e7c92bfcff24d40.png"},{"id":99790443,"identity":"fc94c658-2c17-4529-8b9c-26e65b57e3e5","added_by":"auto","created_at":"2026-01-08 12:58:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":93563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFMC63-OKT3 and ZHER-OKT3 promote T cells to eliminate the antigen-expressing tumor cells.\u003c/strong\u003e(A) Schematic illustration of the construction process of FMC63-OKT3. (B) Cytotoxicity assay of the Nalm6 cells incubated with PBS, FMC63-Fc-SC, OKT3-Fc-ST or FMC63-OKT3 in the presence of T cells, the effector cells: target cells (E:T = 1:1). The antibody concentration is 1000 ng/ml. (C) Nalm6 cells were co-cultured with T cells at an E:T ratio of 1:1 in the presence of the indicated concentrations of FMC63-OKT3 for 24 h. (D) Quantification of activation markers in T cells after incubation with Nalm6 cells, and plus PBS, FMC63-Fc-SC, OKT3-Fc-ST or FMC63-OKT3 for 24 h. The antibody concentration is 1000 ng/ml. (E) T cells were co-cultured with Nalm6 cells for 24 hours in the presence or absence of FMC63-OKT3, and cytokine levels in the culture supernatant were quantified by ELISA. (F)Schematic illustration of the construction process of ZHER-OKT3. (G) Cytotoxicity assay of the Daoy cells incubated with PBS, ZHER-SC, OKT3-Fc-ST or ZHER-OKT3 in the presence of T cells, the effector cells: target cells (E:T = 1:1). The antibody concentration is 1000 ng/ml. (H) Daoy cells were co-cultured with T cells at an E:T ratio of 1:1 in the presence of the indicated concentrations of ZHER-OKT3 for 24 h. (I) Quantification of activation markers in T cells after incubation with Daoy cells, and plus PBS, ZHER-SC, OKT3-Fc-ST or ZHER-OKT3 for 24 h. The antibody concentration is 1000 ng/ml. (J) T cells were co-cultured with Daoy cells for 24 hours in the presence or absence of ZHER-OKT3, and cytokine levels in the culture supernatant were quantified by ELISA. All cytotoxicity assays were assessed by flow cytometry for DAPI staining, and all data are presented as means ± s.d., n = 3.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/626c6db1484a963d1dbbc45d.png"},{"id":99496359,"identity":"af08a94c-3afe-4fde-b9bf-3251bfaa4c3a","added_by":"auto","created_at":"2026-01-05 06:29:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eaEGFR-OKT3 and aPDL1-OKT3 mediates T cells to kill the antigen-expressing tumor cells.\u003c/strong\u003e (A) Binding of aEGFR-SC and aPDL1-SC with EGFR/PDL1-positive Daoy cells and EGFR/PDL1-negative Nalm6 cells was detected by flow cytometry. (B)Schematic illustration of the construction process of aEGFR-OKT3 and aPDL1-OKT3. (C and D) T cell cytotoxicity was analyzed following a 24-hour co-culture with Daoy cells (E:T=1:1), treated with either EGFR-OKT3(C) or PDL1-OKT3(D). (E and F) Daoy cells were co-cultured with T cells at an E:T ratio of 1:1 in the presence of the indicated concentrations of EGFR-OKT3(E) or PDL1-OKT3(F) for 24 h. (G and H) T cells were co-cultured with Daoy cells for 24 hours in the presence or absence of EGFR-OKT3(G) or PDL1-OKT3(H), and cytokine levels in the culture supernatant were quantified by ELISA. All cytotoxicity assays were assessed by flow cytometry for DAPI staining, and all data are presented as means ± s.d., n = 3.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/46bed7348e9416112e41da89.png"},{"id":99790455,"identity":"756e59c1-43ce-4415-ade2-1d8bbd134738","added_by":"auto","created_at":"2026-01-08 12:58:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eaPDL1-OKT3 mediates CAR19-T cells to kill the CD19-expressing tumor cells.\u003c/strong\u003e (A) Structure of CAR19. (B) The expression levels of CAR were analyzed by flow cytometry. Ctrl indicates untransduced T cells. (C) Flow cytometry analysis of PDL1 expression in PDL1-overexpressing Nalm6 cells. (D) CAR19-T and Nalm6-PDL1 cells were co-cultured for 24 h at different E:T ratios, with or without 1000 ng/mL aPDL1-OKT3 (left).T cells or CAR19-T cells were co-cultured with Nalm6-PDL1 cells at E:T as 1:10 for 24 h with or without 1000 ng/ml aPDL1-OKT3(right). Cell lysis ratio was analyzed by flow cytometry. (E) T cells or CAR19-T cells were co-cultured with Nalm6-PDL1 cells at E:T as 1:10 for 24 h with or without 1000 ng/ml aPDL1-OKT3; PBS as control. The ratio of CD69+ or CD25+ cells of CD8+ T cells was assessed by flow cytometry. (F) Following a 48-hour stimulation with CD3/CD28 antibodies (5 μg/mL), the ratios of CD4\u003csup\u003e+ \u003c/sup\u003eand CD8\u003csup\u003e+\u003c/sup\u003eT cells(down) and CAR expression levels(top) of CAR-T cells. (G) Following a 48-hour stimulation with CD3/CD28 antibodies (5 μg/mL), the PD1 expression levels of CAR-T cells. (H) CAR19-T or activated CAR19-T cells were co-cultured with Nalm6-PDL1 cells at E:T as 1:10 for 24 h with or without 1000 ng/ml aPDL1-OKT3. (I) The expression levels of CAR were analyzed by flow cytometry. Ctrl indicates untransduced T cells. (J) T cells or B7H3 CAR-T cells were co-cultured with Daoy cells at E:T as 1:5 for 24 h with or without 1000 ng/ml aEGFR-OKT3. Cell lysis ratio was analyzed by flow cytometry. (K) T cells or B7H3 CAR-T cells were co-cultured with Daoy cells at E:T as 1:5 for 24 h with or without 1000 ng/ml aEGFR-OKT3; PBS as control. The ratio of CD69+ cells of T cells was assessed by flow cytometry.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/80b9659c11b33c9e74842479.png"},{"id":99802908,"identity":"3885e7ee-e6db-4030-8e76-2f500c112f68","added_by":"auto","created_at":"2026-01-08 14:09:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1220999,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/23a3cff9-4a73-4fc0-b195-13e147b8e60b.pdf"},{"id":99496364,"identity":"0dcd4290-1ac7-49eb-88fe-d152fbc459e3","added_by":"auto","created_at":"2026-01-05 06:29:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":316697,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/af899db0d2513bc5a52184f6.docx"},{"id":99790872,"identity":"e817a03b-3249-4c0e-b83b-9d9c992351a2","added_by":"auto","created_at":"2026-01-08 12:58:48","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8371642/v1/52981eafec82f600353518ec.png"}],"financialInterests":"","formattedTitle":"Modular construction of bispecific antibodies through bioconjugation for T cell-based immunotherapy","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBispecific antibodies (bsAbs) have emerged as a highly promising class of therapeutic agents, introducing revolutionary changes to the fields of medicine and biotechnology\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The unique molecules, engineered to bind two distinct antigens simultaneously\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, function by bridging tumor-associated antigens (TAAs) and immune effector cells such as T cells. BsAbs address the limitations of monospecific antibodies, enabling major histocompatibility complex-independent redirection of cytotoxicity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Such a dual-targeting strategy has demonstrated significant clinical efficacy in hematological malignancies; for instance, blinatumomab (anti-CD19/CD3) achieves a 43% complete remission rate in relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The modular nature of bsAbs further supports the construction of customized immune synapses, establishing their position as a core tool in next-generation immuno-oncology\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This dual-targeting feature opens up broad potential medical applications, particularly in the treatment of complex diseases such as cancer and autoimmune disorders\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the structural complexity and manufacturing of bispecific antibodies present significant challenges.\u003c/p\u003e \u003cp\u003eThe unnatural chain combinations frequently lead to mispairing, aggregation, and low expression yield, imposing stringent requirements on manufacturing processes and quality control\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Current development efforts are increasingly focusing on optimizing designs to enhance safety, improve pharmacokinetic properties, and expand therapeutic indications\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Through development of more stable humanized frameworks and implementation of modular assembly platforms, bispecific antibodies could assume a more central role in the current era of precision medicine, ultimately fulfilling their considerable therapeutic potential\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe combined application of CAR-T (Chimeric Antigen Receptor T cells) cells and bispecific antibodies is a promising strategy in cancer immunotherapy. CAR-T cells have demonstrated considerable efficacy in the treatment of certain hematological malignancies\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e; however, their efficacy in solid tumors is still limited by antigen heterogeneity\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, immunosuppressive microenvironments\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and on-target/off-tumor toxicity\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. To enhance CAR-T function, a strategy integrating BsAbs has emerged\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. BsAbs can recruit endogenous T cells to CAR-T-resistant tumor subsets or target alternative TAAs\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, thereby expanding antigen coverage and reducing immune escape\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This further improves the specificity and efficacy of CAR-T cell-mediated killing\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Preclinical studies have shown that anti-CD20/CD3 BsAbs can enhance the efficacy of CD19 CAR-T cells in heterogeneous lymphoma models significantly\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, highlighting the potential of the integrated treatment platform.\u003c/p\u003e \u003cp\u003eTraditional BsAb production is confronted with scalability, stability, and homogeneity challenges. The SpyCatcher-SpyTag system is a powerful protein-ligation technology with numerous applications in protein engineering and biotechnology\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It is based on a modified domain from a \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e surface protein, SpyCatcher, which specifically recognizes a cognate 13 - amino - acid peptide, SpyTag\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Upon recognition, an irreversible covalent isopeptide bond forms between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The reaction occurs rapidly and efficiently under physiological conditions, and is insensitive to numerous factors such as buffer composition, temperature, and pH\u003csup\u003e28\u003c/sup\u003e. The SpyCatcher-SpyTag system offers several advantages over traditional protein-conjugation methods\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Formation of a covalent bond ensures a stable and robust linkage between proteins, which is crucial for applications such as construction of multi-protein complexes or modification of proteins with functional moieties\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In addition, the small size of the SpyTag peptide (only 13 amino acids) minimizes potential interference with the structure and target protein function. Furthermore, the system is highly versatile and can be used to conjugate a wide range of proteins, both in vitro and in vivo\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe aim of the this study was to modularly construct various bispecific antibodies to effectively activate T cells or CAR-T cells and mediate their killing effect on tumor cells. The use of the SpyCatcher-SpyTag system allows for precise and efficient construction of the bispecific antibodies, which is expected to address some challenges associated with traditional bispecific antibody preparation methods. Bispecific antibody was used to conjugate an antibody fusion protein targeted tumor with SpyCatcher to an antibody fusion protein incorporating the anti-CD3 domain (7G03 and OKT3) and SpyTag. BsAbs targeting HER2, CD19, PDL1, and EGFR were generated by constructing ZHER (anti-HER2), FMC63 (anti-CD19), anti-PDL1, and anti-EGFR bispecific antibodies, respectively, and the bsAbs were applied to target tumor cells expressing CD19, PDL1, or EGFR. Furthermore, modular bioconjugated construction of bispecific antibodies was used to enhance CAR-T cell cytotoxicity. Our research could not only contribute to the development of novel immunotherapeutic strategies but also provide insights that could facilitate the application of the SpyCatcher-SpyTag system in antibody engineering.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eModular Construction and Cytotoxic Efficacy of the Bispecific Antibodies\u003c/h2\u003e \u003cp\u003eTo target human T cells, an anti-CD3 nanobody fused with SpyTag, named 7G03-SpyTag (7G03-ST), was designed. To achieve efficient site-specific conjugation via the SpyCatcher-SpyTag system, an anti-CD19 scFv-Fc antibody fused with SpyCatcher was expressed and purified, and termed FMC63-Fc-SpyCatcher (FMC63-Fc-SC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Since SpyCatcher and SpyTag spontaneously recognize and form a stable covalent bond under conventional conditions, the SpyTag-modified 7G03 could be site-specifically conjugated with FMC63-Fc-SC upon mixing, yielding the modular bispecific antibody FMC63-7G03 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). To obtain and cultivate T cells, T cells were isolated from healthy donors and stimulated in vitro with CD3/CD28 antibodies (5 \u0026micro;g/mL) for 48 h. To evaluate the T cell-mediated cytotoxicity induced by FMC63-7G03, the activated T cells were then co-cultured with Nalm6 cells for 24 h with bsAbs. Cytotoxicity against Nalm6 cells was assessed by flow cytometry. The results demonstrated that FMC63-7G03 effectively redirected activated T cells to kill Nalm6 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In contrast, FMC63-7G03 alone did not affect the Nalm6 cells viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, the expression of T cell activation markers (CD25 and CD69) was examined following co-culture with Nalm6 cells in the presence or absence of specific antibodies. Significant upregulation of CD25 and CD69 was observed only when FMC63-7G03 was present (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, FigS.1A). Additionally, after 24 h of co-culture with tumor cells in the presence of FMC63-7G03, ELISA analysis revealed a marked increase in the secretion of effector cytokines\u0026mdash;including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2\u0026mdash;in the culture supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The FMC63-7G03 bispecific antibody, successfully constructed using the nanobody 7G03-ST, can direct T cells to eliminate CD19-expressing Leukemia cells. The findings indicate that modularly constructed bispecific antibodies could effectively activate T cells and direct them to specifically eliminate antigen-expressing tumor cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModular Constructed Bispecific Antibodies Mediates T Cell Killing of Solid Tumor Cells\u003c/h3\u003e\n\u003cp\u003eTo investigate whether the modular approach could be extended to other targets, the anti-HER2 ZHER-SpyCatcher (ZHER-SC) protein, an affibody antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and this was site-specifically conjugated with 7G03-ST to form the modular bispecific antibody ZHER-7G03 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). To assess whether ZHER-7G03 could redirect T cells to kill antigen-matched tumor cells, activated T cells were co-cultured with Daoy (HER2\u003csup\u003e+\u003c/sup\u003e) target cells at an effector-to-target (E:T) ratio of 1:1 in the presence of 1000 ng/mL ZHER-7G03 for 24 h. Target cell death was quantified by flow cytometry, revealing specific lysis of approximately 60% of the cells in the ZHER-7G03 group, which was significantly higher than those in the control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Incubation with ZHER-7G03 alone did not affect tumor cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, T cells treated with ZHER-7G03 exhibited significantly elevated surface expression of the activation markers CD25 and CD69 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, FigS. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). ELISA analysis of culture supernatants also demonstrated markedly increased levels of the effector cytokines TNF-α, IFN-γ, and IL-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The results indicate that ZHER-7G03, constructed via the SpyCatcher-SpyTag system, is biologically active. It not only induces antigen-specific T cell activation but also promotes secretion of cytotoxic effector molecules, enabling precise elimination of HER2\u003csup\u003e+\u003c/sup\u003e solid tumor cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eValidation of Versatility and Tumor Killing Efficacy of the Modular Bispecific Antibody Construction Platform\u003c/h3\u003e\n\u003cp\u003eTo validate the versatility of the modular bispecific antibody architecture presented, 7G03-ST was replaced with OKT3-Fc-SpyTag (OKT3-Fc-ST), an scFv-Fc fusion antibody that also targets CD3 to improve stability by Fc segments. OKT3-Fc-ST was efficiently and stably conjugated with the scFv antibody FMC63-Fc-SC and the affibody ZHER-SC to generate FMC63-OKT3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Figs. S3A) and ZHER-OKT3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, Figs. S3E). Pre-stimulated T cells were co-cultured with target cells at a 1:1 ratio for 24 h, and the results indicated that OKT3-Fc-ST alone could activate T cells moderately; however, in the presence of FMC63-OKT3 or ZHER-OKT3, T cell-mediated cytotoxicity was enhanced significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, G, Figs. S3B, F). Neither bispecific antibody affected tumor cell viability alone (Figs. S3C, G). Tumor cell killing exhibited a dose-dependent response to concentration of bispecific antibodies in the system (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, H). Moreover, T cells treated with the bispecific antibodies exhibited significantly elevated expression of the activation markers CD25 and CD69 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, I, Figs. S3D, H). ELISA analysis of culture supernatants revealed markedly increased levels of effector cytokines\u0026mdash;TNF-α, IFN-γ, and IL-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, anti-EGFR and anti-PDL1 nanobodies fused with SpyCatcher: aEGFR-SC and aPDL1-SC were constructed. Both nanobodies bound specifically to Daoy cells expressing their respective targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). They were conjugated with OKT3-Fc-ST to form aEGFR-OKT3 and aPDL1-OKT3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Figs. S4A, B). Neither bispecific antibody influenced tumor cell growth alone (Figs. S4E, F), yet both effectively redirected T cells to kill target cells in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D, Figs. S4C, D). Additionally, they promoted T cell activation and effector cytokine secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;H, Figs. S4G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results confirm the broad versatility of the modular bispecific antibody construction platform based on the SpyCatcher-SpyTag system. By modular assembly of CD3-targeting OKT3-Fc-ST with various targeting moieties\u0026mdash;including FMC63, ZHER, aEGFR, and aPDL1\u0026mdash;multiple bispecific antibodies were constructed successfully, each effectively mediating T cell killing of antigen-matched target cells. The platform provides a reliable and flexible strategy for rapid generation of diverse bispecific antibodies.\u003c/p\u003e\n\u003ch3\u003eModular Construction of Bispecific Antibodies through Bioconjugation Enhances CAR-T Cell-Mediated Cytotoxicity\u003c/h3\u003e\n\u003cp\u003eTo determine whether modular bispecific antibodies can enhance CAR-T cell cytotoxicity, second-generation CD19-specific CAR-T cells incorporating 4-1BB as a costimulatory domain were generated. The CD19 CAR construct consisted of an extracellular anti-CD19 (FMC63) single-chain variable fragment (scFv), a transmembrane domain, and intracellular signaling domains derived from human 4-1BB and CD3ζ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After 48-h stimulation, T cells were transduced with a CD19 CAR lentiviral vector, resulting in approximately 60% CAR-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, Nalm6 tumor cells were engineered to overexpress PDL1 (Nalm6-PDL1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). When T cells or CAR-T cells were co-cultured with Nalm6-PDL1 for 24 h, aPDL1-OKT3 further activated CD8\u003csup\u003e+\u003c/sup\u003e CAR-T cells and enhanced their tumor-killing capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine T cell activation dynamics, CAR-T cells were restimulated with CD3/CD28 antibodies for 48 h. This not only increased the proportion of CD8\u003csup\u003e+\u003c/sup\u003e CAR-T cells and CAR positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) but also induced PD-1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), indicating emergence of a pre-exhaustion phenotype in a subset of T cells, following strong activation. Subsequently, the CAR-T cells were co-cultured with Nalm6-PDL1 cells for 24 h. Although the cytotoxic efficacy of CAR-T cells was reduced partially due to the exhausted phenotype, the presence of aPDL1-OKT3 effectively restored T cell-mediated tumor killing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In addition, second-generation B7H3-specific CAR-T cells were established, also containing the 4-1BB costimulatory domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Co-culture of T cells or CAR-T cells with Daoy cells for 24 h demonstrated that aEGFR-OKT3 similarly enhanced CAR-T cell activation and tumor cell killing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ, K). The observations suggest that modular construction of bispecific antibodies through bioconjugation can partially overcome or compensate for functional impairments in early-exhausted CAR-T cells.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn the present study, an scfv fusion protein incorporating FMC63 (anti-CD19) and SpyCatcher domains was initially expressed, and the FMC63 domain specifically targets the CD19 antigen, whereas the SpyCatcher domain enables conjugation with the nanobody 7G03-ST, thereby directing T cells to recognize and eliminate CD19-positive tumor cells. Furthermore, additional bispecific antibodies were generated by expressing fusion proteins containing either an anti-HER2 affibody, an anti-PDL1 nanobody, or an anti-EGFR nanobody, which were site-specifically conjugated with 7G03-ST or OKT3-ST. In vitro cytotoxicity assays demonstrated that all the bispecific antibodies effectively mediated T cell-induced tumor cell killing. Notably, the bispecific antibody aPDL1-OKT3 not only enhanced CAR19-T cell-mediated cytotoxicity against Nalm6-PDL1 tumor cells but also restored the functional capacity of early-exhausted CAR-T cells partially.\u003c/p\u003e \u003cp\u003eCollectively, the findings indicate that modular proteins constructed via the SpyCatcher/SpyTag system can effectively bridge T cells and tumor cells through a bispecific binding mode. Such interaction induces antigen-specific T cell activation and promotes secretion of cytotoxic effector molecules, leading to precise tumor cell elimination. The modular design strategy, leveraging efficient and stable site-specific conjugation, establishes a transferable technological paradigm for engineering bispecific antibodies. Its versatility demonstrates considerable potential in translational immunotherapy applications across diverse antigen targets.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eReagents and Materials\u003c/h2\u003e \u003cp\u003ePhosphate buffered saline (PBS), Isopropyl β-d-1-thiogalactopyranoside(IPTG), Kanamycin B, imidazole, Coomassie brilliant blue, and Ni-IDA-Sefinose resin were purchased from Sangon (Shanghai, China). DAPI was obtained from Beyotime Biotechnology (Jiangsu, China). MaxFB, MaxFA6, and HEK293 MaxD media were purchased from MediumBank Biotechnology Co., Ltd. (Shanghai, China). Flow cytometry antibodies and dyes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Minimum Essential Medium (MEM), MEM Non-Essential Amino Acids Solution (NEAA), Dulbecco\u0026rsquo;s Modified Eagle Medium, RPMI 1640 basic Medium, and Fetal Bovine Serum (FBS) were purchased from Gibco (Frederick, MD, USA). X-VIVO 15 was purchased from Lonza (Basel, Switzerland). Serum-free CELLSAVING and penicillinstreptomycin (P/S) were obtained from NCM Biotech (Suzhou, China). Human CD3 (humanized OKT3) and CD28 antibodies were purchased from Sino Biological Inc. (Beijing, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines and primary cells\u003c/h3\u003e\n\u003cp\u003eCell lines: The human medulloblastoma cell line Daoy and the B-cell acute lymphoblastic leukemia cell line Nalm6 were obtained from the American Type Culture Collection (ATCC). Daoy cells were maintained in MEM medium supplemented with 10% FBS, 1% penicillin\u0026ndash;streptomycin (P/S), and 1% non-essential amino acids (NEAA). Nalm6 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% P/S. The human embryonic kidney epithelial cell line HEK293T was acquired from the National Collection of Authenticated Cell Cultures and grown in HEK293 MaxD medium supplemented with 3% MaxFA6 and 0.3% MaxFB. All cell lines were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e \u003cp\u003ePeripheral Blood Mononuclear Cells and T Cell Isolation: Human primary T cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) obtained from healthy donors. Whole blood was processed by density gradient centrifugation using a human PBMC isolation buffer. The isolated PBMCs were cultured in X-VIVO 15 medium containing 4% FBS and 300 U/mL recombinant human IL‑2 (SL pharm, Beijing, China). T cell activation was induced by a 48-h stimulation with anti-human CD3 and anti-human CD28 antibodies, each at a concentration of 5 \u0026micro;g/mL. Following activation, T cells were maintained in culture for 5\u0026ndash;12 d prior to subsequent experiments.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein Production and Purification\u003c/h2\u003e \u003cp\u003eThe OKT3-Fc-ST construct, comprising OKT3, Fc, SpyTag, and a His-Tag, was cloned into the protein expression vector pSB. Similarly, the FMC63-Fc-SC construct\u0026mdash;consisting of FMC63, Fc, SpyCatcher, and a His-Tag\u0026mdash;was also inserted into the pSB vector. The corresponding nucleotide sequences are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Transfection of the expression vectors into HEK-293T cells was performed using Lipofectamine 3000 transfection reagent (Life Technologies, Carlsbad, CA, USA), and stable cell lines were established under selection with 2 \u0026micro;g/mL puromycin.\u003c/p\u003e \u003cp\u003eAdditionally, 7G03-SpyTag, ZHER-SpyCatcher, aPDL1-SpyCatcher, and aEGFR-SpyCatcher were constructed as fusions of the respective antibody fragments with SpyTag or SpyCatcher and a His-Tag, and were cloned into the pET28A expression vector. The sequences of the constructs are also listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The recombinant vectors were transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e for protein expression. Positive clones were cultured in Luria-Bertani liquid medium supplemented with 10 \u0026micro;g/mL kanamycin at 37\u0026deg;C until the OD600 reached 1.0. Protein expression was induced by adding 1 mM IPTG, followed by overnight incubation at 16\u0026deg;C. Bacterial cells were harvested by centrifugation at 4000 \u0026times;g for 5 min and lysed via ultrasonication. The soluble fraction was obtained by centrifugation at 12,000 \u0026times;g for 30 min at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eProtein purification was performed using Ni-IDA gravity chromatography columns. Culture supernatants from HEK-293T cells or bacterial lysates were centrifuged at 4\u0026deg;C (4000 \u0026times;g, 5 min) and applied to the columns. After washing with 20 or 40 mM imidazole in PBS, the target proteins were eluted using 200 or 500 mM imidazole in PBS. Elution fractions were analyzed by SDS-PAGE with Coomassie blue staining, and those showing the expected protein bands were pooled. Proteins were concentrated and buffer-exchanged into PBS using an Amicon Ultra-4 10 kDa centrifugal filter device (Merck, Rahway, NJ, USA) with three dialysis steps. Purified proteins were aliquoted and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for long-term storage or at 4\u0026deg;C for immediate use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSite-Specific Ligation of SpyCatcher‑Tagged Proteins with SpyTag‑Containing Constructs\u003c/h2\u003e \u003cp\u003eThe tumor-targeting protein fused to SpyCatcher was mixed with either 7G03-SpyTag or OKT3-Fc-SpyTag at a 1:1 molar ratio (SpyTag:SpyCatcher). The reaction was carried out with gentle rotation at 37\u0026deg;C for 2 h, followed by further incubation overnight at 4\u0026deg;C under continuous gentle rotation. To confirm formation of the conjugate, the products were analyzed by SDS‑PAGE with Coomassie blue staining, and samples exhibiting the expected molecular weight bands were selected for subsequent use.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro Cytotoxicity Study.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTumor cells were first labeled with 2.5 \u0026micro;M Cell Proliferation Dye (CPD) or 0.25 \u0026micro;M CFSE for 15 min at 37\u0026deg;C. Subsequently, T cells and labeled tumor cells were co-cultured in 48-well plates at a 1:1 effector-to-target (E:T) ratio. Phosphate-buffered saline (PBS), modular bispecific antibodies (bsAbs), or corresponding controls were then administered, and the plates were incubated at 37\u0026deg;C for 24 h. Following incubation, cells were stained with 5 \u0026micro;g/mL 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) for 15 min at 4\u0026deg;C, washed once with PBS, and subjected to flow cytometric analysis. Tumor cell lysis was determined by quantifying the populations of CPD⁺/DAPI⁺ or CFSE⁺/DAPI⁺ cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of CAR-T cells\u003c/h2\u003e \u003cp\u003eHEK-293T cells were seeded in 100-mm cell culture dishes and cultured overnight. The cells were then co-transfected with CAR19 or CARB7H3 lentiviral vectors, along with the packaging plasmids psPAX2 and pMD2.G, using a liposome-based transfection reagent. Viral supernatant was collected at 48 and 72 h post-transfection and concentrated using polyethylene glycol 8000 (PEG8000). The resulting viral precipitate was resuspended in phosphate-buffered saline (PBS). For T cell transduction, primary human T cells were stimulated with anti-human CD3 and CD28 antibodies for 48 h, followed by exposure to the respective lentiviral particles in the presence of 10 \u0026micro;g/mL polybrene. Transduction efficiency was evaluated 48 h later by flow cytometry based on the percentage of CAR-positive cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eTwo-tailed unpaired t-tests were used to compare two groups. One-way Analysis of Variance was used to compare three or more groups in a single condition. All values in the study were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). Differences with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*) P\u0026thinsp;\u0026lt;\u0026thinsp;0.01(**) P\u0026thinsp;\u0026lt;\u0026thinsp;0.001(***), and **** P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001(****) were considered significant\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eASSOCIATED CONTENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAdditional data, including the\u0026nbsp;sequences of protein utilized in this work\u0026nbsp;and additional experimental results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.-Q.S, L.W., and J.X. designed and performed the experiments, interpreted the results, and prepared the manuscript. S.L. and\u0026nbsp;J.-M.Z performed some of the experiments. K.-M.C. and C.-W.D. conceived of the study, designed experiments, supervised research, and wrote the manuscript. S.-Q.S, L.W., and J.X. contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Key R\u0026amp;D Program of China (NO. 2021YFA1100800 to A.-B.L.), the Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China (Grant No. 2023ZQNZD012) and the Natural Science Foundation of Fujian Province (No. 2024J011122).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Pediatric Translational Medicine Institute of Shanghai Children\u0026rsquo;s Medical Center for technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e Permission from the Institutional Animal Ethical Committee was received before making these experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e All data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHolliger, P., \u0026amp; Hudson, P. 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Power to the protein: enhancing and combining activities using the Spy toolbox. \u003cem\u003eChemical Science\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(28), 7281\u0026ndash;7291.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bispecific antibodies, SpyCatcher-SpyTag system, T cell-based immunotherapy, CAR-T cells, Site-specific Conjugation.","lastPublishedDoi":"10.21203/rs.3.rs-8371642/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8371642/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBispecific antibodies (bsAbs) represent a groundbreaking advance in antibody engineering, overcoming therapeutic limitations of monoclonal antibodies through dual-targeting capabilities. However, their clinical translation is often hindered by structural and manufacturing complexities. In this study, we developed a modular bsAb platform utilizing the SpyCatcher-SpyTag site-specific coupling system for T cell-based immunotherapy. We successfully assembled functional bsAbs by conjugating tumor-targeting SpyCatcher-fused antibodies (anti-CD19, FMC63-Fc-SC or anti-HER2, ZHER-SC) with SpyTag-fused anti-CD3 domains (7G03-ST). These bsAbs effectively killed CD19-expressing leukemic cells and HER2-positive solid tumor cells. Furthermore, through substituting the anti-CD3 domain (e.g., OKT3-ST), we readily generated bsAbs targeting CD19, HER2, PD-L1 and EGFR, all of which specifically engaged their respective tumor antigens. More importantly, bsAbs constructed via this modular strategy significantly enhanced the cytotoxicity of CAR-T cells. In conclusion, this flexible bioconjugation platform offers a reliable and efficient technical solution for the rapid development of diverse bispecific antibodies.\u003c/p\u003e","manuscriptTitle":"Modular construction of bispecific antibodies through bioconjugation for T cell-based immunotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 06:29:09","doi":"10.21203/rs.3.rs-8371642/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f6913a7-0552-48b7-acb7-c6312c0f8644","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T14:45:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-05 06:29:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8371642","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8371642","identity":"rs-8371642","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}