Functional identification of the C-terminal domain of rhCNB

Functional identification of the C-terminal domain of rhCNB | 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 Functional identification of the C-terminal domain of rhCNB Ziwei Zhu, Li Tong, Hongcui Ma, Huinan Yang, Jinju Yang, Qun Wei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7943596/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 Recombinant human Calcineurin B subunit (rhCNB) has emerged as a promising antitumour candidate. Its antitumour activity is mediated by the secretion of proinflammatory cytokines and chemokines from both innate and adaptive immune cells, thereby enhancing their antigen-presenting capacity. Moreover, rhCNB is rapidly internalized by various tumour cells, demonstrating specific tumour-targeting properties. Previous studies have revealed that the C-terminal domain is responsible for the internalization and targeting ability of rhCNB; however, the precise antitumour functional domain remains unexplored. To address this, we engineered a truncated variant (designated DC, comprising amino acids 85–169 of rhCNB). Functional characterization revealed that DC promoted the maturation and differentiation of bone marrow-derived dendritic cells (BMDCs), stimulated the production of cytokines by BMDCs, were internalized into tumour cells, accumulated at tumour sites, and synergistically enhanced the tumoricidal activity of paclitaxel. Administration of the same dose (5 mg/kg) of DC and rhCNB to H22 tumour-bearing mice resulted in tumour growth inhibition rates of 40.5% and 53.73%, respectively, with no statistically significant difference between the two treatments. Collectively, these findings identify DC as the core functional domain responsible for the antitumour effects of rhCNB. rhCNB tumour therapy immunostimulation targeting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The calmodulin phosphatase B subunit (CNB) is the regulatory subunit of calmodulin phosphatase (CN), and its main function in vivo is to regulate the activity of calmodulin phosphatase A subunit (CNA) [ 1 , 2 ]. Studies have indicated that CNB has other physiological functions independent of CNA; for example, CNB deficiency is associated with a high risk of squamous cell carcinoma [ 3 ]. Research in our laboratory revealed that recombinant CNB significantly inhibited tumour growth and prolonged survival in various mouse models [ 4 , 5 ]. Further studies indicated that it promoted dendritic cell maturation and antigen presentation, increased the phagocytic activity of macrophages and natural killer (NK) cells, and triggered the secretion of proinflammatory cytokines and chemokines. These properties enable its use as both a tumour vaccine adjuvant and an immunomodulator, effectively inducing cellular and humoral immunity [ 6 , 7 ]. Mammalian CNB is composed of 169 amino acids and has 4 EF-hand structures, belonging to the calcium-binding protein family. The typical structural feature of the EF hand is a spiral structure, and each EF hand structure can bind to a calcium ion. Different EF-hand regions exhibit vastly different binding affinities for calcium ions, varying by up to a million-fold [ 8 ]. The main segments mediating the internalization and targeting of rhCNB are located at the C-terminus [ 9 ]; however, it is not known if the main segment mediating the antitumour and immune activation functions is concentrated at the C-terminus. To explore the main antitumour functional segments of rhCNB, we prepared a C-terminal fragment of rhCNB (amino acids 85–169) and characterized its function. The results indicated that the C-terminus of rhCNB is the key domain responsible for antitumour activity. 2. Methods 2.1. Cell lines and animal culture The cell lines used in this study included the human hepatocellular carcinoma cell lines SMMC-7721, HepG2, and MHCC-97H; the mouse hepatocellular carcinoma cell line H22; and the mouse colon carcinoma cell line CT26. SMMC-7721, HepG2, H22, and CT26 cells were purchased from ATCC, and MHCC-97H cells were obtained from the Shanghai Institute of Cell Biology. These cell lines were confirmed to be free of Mycoplasma contamination through PCR and fluorescence staining (Fig. S1 ). Female ICR (8 weeks old, weighing 30–40 g) and BALB/c nude mice (6–8 weeks old, weighing 20–22 g) were used in this study. These mice were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China). 2.2. Construction, Expression and Purification of DC The DC fragment was obtained from the rhCNB (P63098) plasmid and constructed by restriction enzyme digestion and ligation. The plasmids containing the correct inserts were transformed into E. coli BL21(DE3) competent cells for protein expression. Seven individual transformants were induced by IPTG for high-expression strain screening, and the expression products were identified by SDS‒PAGE and Western blot analysis. The selected high-expression strain was expanded for large-scale protein production. The purification of DC was performed using the method previously established for rhCNB. Briefly, the bacterial cells were harvested and resuspended in Buffer A (pH 7.4, 20 mM Tris-HCl). The suspension was subsequently sonicated, boiled in a water bath for 20 minutes, and centrifuged at 10,000 rpm for 20 minutes to collect the supernatant. The supernatant containing recombinant DC protein was loaded on phenyl hydrophobic chromatography equilibrated with Buffer B (20 mM Tris-HCl, 2 mM CaCl 2 , and 0.5 M NaCl, pH 7.4), and the DC protein was subsequently eluted with Buffer C (20 mM Tris-HCl, 0.5 mM EGTA, pH 7.4). The eluted fraction from the phenyl column was lyophilized and subjected to buffer exchange into 20 mM phosphate buffer (PB, pH 7.4) using a Sephadex G25 desalting column. The collected fraction was further purified by DEAE ion-exchange chromatography, which was primarily employed for endotoxin removal. The sample was loaded in Buffer D (20 mM PB, pH 7.4) and eluted with Buffer E (20 mM PB, 100 mM NaCl, pH 7.4). Finally, the purified fraction was concentrated by freeze-drying. The concentration of the final product was measured by a BCA assay, the endotoxin content was detected using limulus reagent, the purity was analysed by SDS‒PAGE, and the activity was determined by the pNPP method. 2.4. DC activity determination The activity of the purified DC was determined by the pNPP (p-nitrophenyl phosphate) method, a simple, widely used colorimetric assay for measuring phosphatase activity. Briefly, enzyme dilutions or diluted CNA were separately added to their corresponding tubes and precooled on ice for 5 min. Subsequently, the activity assay solution, either alone or with purified DC or rhCNB, was introduced, and the mixtures were incubated at 30°C for 20 min. The reactions were stopped, and the absorbance at 410 nm (OD₄₁₀) was recorded. Each treatment was performed with three technical replicates, and the entire experiment was independently repeated three times. In this setup, CNA alone served as the negative control, CNA + rhCNB as the positive control, enzyme dilution alone as the blank control, and CNA combined with DC (CNA + DC) constituted the test group. 2.5. Cellular Uptake of Exogenous DC 5-FAM-labelled rhCNB and DC were prepared for cellular uptake analysis. Briefly, an equimolar amount of 5-FAM was mixed with either DC or rhCNB and incubated in the dark at room temperature for 1 h. The mixture was dialyzed overnight at 4°C against PBS using a 3.5 kDa cut-off dialysis membrane to remove unbound dye. Cells (2 × 10⁵) seeded in 60 mm Petri dishes were treated with 5 µM 5-FAM-labelled rhCNB, 5-FAM-labelled DC, or unlabelled DC (negative control) and incubated at 37°C for 30 minutes. The cells were subsequently washed three times with PBS, exposed to acid-stripping buffer (pH 5.0 Gly-HCl buffer), and fixed with 4% paraformaldehyde. Cellular uptake was visualized using a Zeiss confocal fluorescence microscope, and fluorescence intensity was quantified with ImageJ software by measuring the mean grey value of the images. 2.6. In Vivo Imaging DC or rhCNB was labelled with Cy7-SE following the procedure described in Section 2.5 . SMMC-7721 tumour-bearing mice or H22 tumour-bearing mice were established by subcutaneously injecting 1×10⁶ cells into the right armpit. When the tumour volume reached approximately 100 mm³, the mice were randomly assigned to one of three groups (n = 9). Each group received a tail vein injection of 100 µg of Cy7-labelled DC, Cy7-labelled rhCNB, or unlabeled DC (as a blank control). In vivo imaging was performed at 6, 8, 10, 24, and 36 h post-injection using a Revvity IVIS Spectrum system. During imaging, mice were anesthetized with isoflurane (2–3% for induction and 1–2% for maintenance) in oxygen. At 6, 24, and 36 h post-injection, the mice were deeply anesthetized with isoflurane followed by cervical dislocation for euthanasia. The tumors and major organs (heart, liver, spleen, lung, and kidney) were collected for ex vivo fluorescence imaging. The fluorescence intensity of the excised tumors was quantified using ImageJ software by measuring the mean RGB values of the images. 2.7. Isolation and Induction of Mouse Bone Marrow-Derived Dendritic Cells Bone marrow cells were isolated from ICR mice by flushing the bone marrow cavity with RPMI-1640 medium. The cell suspension was gently dispersed, filtered through a 200-mesh nylon sieve, and centrifuged at 1500 × g for 5 min. After two washes with PBS, the cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 25 ng/mL recombinant mGM-CSF and 25 ng/mL mIL-4. The medium was replaced every two days, and CD11c staining was performed every three days to monitor differentiation. On Day 6, nonadherent and loosely adherent cells were collected as immature dendritic cells (imDCs). For stimulation, imDCs were seeded in 12-well plates at 1×10⁶ cells/well. The following day, the cells were treated with 100 µg/mL rhCNB, 100 µg/mL DC, or PBS (as a vehicle control) and coincubated for 48 h. The expression of the surface costimulatory molecules CD80 and CD86 was subsequently analysed by flow cytometry (FCM), and the levels of cytokines and chemokines in the supernatants were determined using commercial ELISA kits. 2.8. MTT Assay for Cell Proliferation MHCC-97H, HepG2, CT26, and SMMC-7721 cells were seeded into 96-well plates at 4000 cells/well. On the following day, the culture medium was replaced with fresh medium containing either rhCNB or DC (at concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.156, or 0 µM), PTX (at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, or 0 µM), or combinations of DC and PTX or rhCNB and PTX. The combination treatments consisted of fixed ratios of rhCNB (or DC) and PTX (5 µM + 1 µM), (2.5 µM + 0.5 µM), (1.25 µM + 0.25 µM), (0.625 µM + 0.0625 µM), (0.3125 µM + 0.03125 µM), (0.156 µM + 0.0156 µM), and (0 µM + 0 µM). After the compounds were added, the cells were incubated for 48 h. Cell proliferation was assessed using a modified MTT assay. 2.9. Tumour Treatment ICR mice (6–8 weeks old, body weight 30–40 g) were randomly divided into 3 groups (n = 10) and received daily intraperitoneal injections (0.2 mL) of normal saline, 5 mg/kg rhCNB, or 5 mg/kg DC. After three administrations, 2 × 10 5 H22 cells were transplanted into the right armpits of ICR mice. Tumour volumes were measured using Vernier callipers every 2 days beginning on Day 5 after tumour challenge. After 25 days, Mice were deeply anesthetized with isoflurane followed by cervical dislocation, and the tumours were removed and weighed. 2.10. Statistical Analysis Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test in GraphPad Prism 6. A P value of < 0.05 was considered to indicate statistical significance, and the significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. 3. Results 3.1. Construction and Purification of the Truncated DC Protein We engineered a truncated variant of rhCNB, designated DC, which consists of the C-terminal region (aa 85–169) and corresponds to half of the full-length rhCNB sequence (Fig. 1 A, green). RhCNB contains four EF-hand motifs; however, the DC variant retains only the two C-terminal motifs (EF-hands 3 and 4), which are critical for its function (Fig. 1 B). SDS‒PAGE and Western blot analyses of IPTG-induced DC transformant colonies confirmed the successful expression of the DC protein. The recombinant protein had the expected molecular weight of approximately 10 kDa and was specifically recognized by an anti-rhCNB polyclonal antibody (Fig. 1 C and 1 D). Clone #4 (marked by a white box in Fig. 1 C), which exhibited optimal expression characteristics, was selected as the production strain. The selected production strain (Clone #4) was subjected to large-scale cultivation. Following cell lysis via sonication, the DC protein was detected primarily in the supernatant (Fig. 1 F, Lanes 2–3). A subsequent heat treatment step effectively enriched the DC protein in the boiled supernatant (Fig. 1 F, Lane 4). This clarified supernatant was then purified through a sequential process of phenyl hydrophobic chromatography, Sephadex G25 buffer exchange, and DEAE ion-exchange chromatography (Fig. 1 E). Analysis of the eluted fractions by SDS–PAGE demonstrated that the phenyl chromatography step significantly increased the purity of the target protein (Fig. 1 F, Lanes 5–8). Buffer exchange using a Sephadex G25 column maintained this high purity (Fig. 1 F, Lanes 9–10). Finally, the DEAE chromatography step yielded a highly pure final product that met the required endotoxin specification of less than 0.1 EU/mg (Fig. 1 F, Lanes 11–12). Western blot analysis confirmed that the purified protein was the target DC variant (Fig. 1 G). CNB, as the regulatory subunit of calcineurin, regulates the activity of the catalytic subunit CNA. To functionally characterize the DC protein, we utilized a pNDD assay to determine whether the DC protein could enhance the catalytic activity of CNA. The results indicated a significant increase in CNA activity by the DC protein (P = 0.0085; Fig. 1 H). These findings verified the successful preparation of the target DC protein, which not only met purity specifications but also retained the intended biological function. 3.2. DC Rapidly Enters Tumour Cells Our previous studies demonstrated that rhCNB can be rapidly internalized into tumour cells. The truncated variant Trun3 (aa 124–169), which was identified as the main segment for rhCNB internalization, can also quickly enter tumour cells and is only slightly weaker than full-length rhCNB. The DC fragment (aa 85–169), which encompasses the complete Trun3 domain, was hypothesized to retain comparable internalization capacity. To detect the effects of DC internalization, labelled DC, rhCNB and unlabelled DC were added to SMMC-7721 cells, and the results of confocal imaging revealed that DC and rhCNB rapidly entered the cells within 15 minutes and that the fluorescence intensities of DC and rhCNB did not significantly differ (Fig. 2 A and Fig. S2). The tumour-targeting and tumour-internalizing abilities of therapeutic proteins are critical for achieving targeted delivery of chemotherapeutic drugs. Such targeted delivery enables chemotherapeutic agents to be transported directly into tumour sites, effectively killing cancer cells while minimizing toxicity to normal organs and tissues, thereby substantially improving therapeutic outcomes. To assess whether DC or rhCNB enhances the cytotoxic effect of chemotherapeutic agents on tumour cells via internalization, we evaluated their combined effects with paclitaxel (PTX) across several tumour cell models. The results revealed that compared with vehicle alone, PTX alone, as well as in combination with DC/PTX and rhCNB/PTX, significantly suppressed tumour cell growth. At higher concentrations, no significant differences were observed among the three treatment groups. However, at lower concentrations, both combination regimens resulted in markedly stronger inhibition than PTX alone did (Fig. 4 B, D, E), whereas no notable difference was detected between the two combination groups. Treatment with rhCNB or DC alone did not inhibit the growth of any of the tumour cell lines tested (Fig. 2 B– 2 E). Further data indicated that both DC and rhCNB significantly increased the cytotoxicity of PTX in the human hepatocellular carcinoma cell lines SMMC-7721 (rhCNB/PTX: P < 0.0001; DC/PTX: P < 0.0001; PTX alone: P = 0.0008; Fig. 2 B) and MHCC-97H (rhCNB/PTX: P = 0.0038; DC/PTX: P = 0.0439; PTX alone: P = 0.0692; Fig. 2 C); HepG2 cells (rhCNB/PTX: P < 0.0001; DC/PTX: P < 0.0001; PTX alone: P = 0.0034; Fig. 2 D), as well as in the mouse colon carcinoma cell line CT26 (rhCNB/PTX: P = 0.0038; DC/PTX: P = 0.0055; PTX alone: P = 0.0692; Fig. 2 E). In summary, these findings suggest that both DC and rhCNB facilitate PTX encapsulation and internalization through their tumour-homing ability, thereby promoting tumour cell apoptosis, lowering the effective dose of PTX needed, and improving its safety profile. 3.3. The DC Protein Also Targets Tumours Previous studies have demonstrated that both exogenous rhCNB and its truncated variant Trun3 exhibit tumour-targeting properties in vivo. In this study, we evaluated the targeting efficacy of DC in SMMC-7721 tumour-bearing BALB/c nude mice and H22 tumour-bearing ICR mice. In SMMC-7721 tumour-bearing mice, both Cy7-labelled DC and Cy7-labelled rhCNB were rapidly distributed and began to accumulate in tumour tissue within 6 hours post-injection, and the signals persisted for up to 24 hours. Notably, fluorescence remained detectable at the tumour site in the Cy7-labelled DC group at the 36-hour time point (Fig. 3 A). Ex vivo fluorescence imaging of the resected tumours further confirmed the retention of both agents within the tumour tissues (Fig. 3 B). Quantitative analysis of tumour fluorescence revealed that compared with the rhCNB group, the DC group exhibited a slightly greater signal intensity at 6 hours, but the difference was not significant (Fig. 3 B). By 24 and 36 hours, the fluorescence intensity in the DC group was markedly greater than that in the rhCNB group, and this difference became statistically significant (24 h: P = 0.0116. 36 h: P = 0.0296. Figure 3 B). In H22 tumour-bearing mice, both labelled proteins markedly accumulated and persisted within tumour tissues (Fig. 3 C and 3 D). At the 6-hour time point, the rhCNB group demonstrated significantly greater fluorescence intensity than the DC group did (P = 0.0359. Figure 3 C). However, this trend was reversed by 24 and 36 hours, at which time the fluorescence of DC was significantly stronger than that of rhCNB (24 h: P = 0.0149. 36 h: P = 0.0404. Figure 3 D). In this study, unlabelled DC served as a blank control. We did not establish a free Cy7 group or a Cy7-labelled unspecific protein group because in a previous study, we detected no significant accumulation of free Cy7 or Cy7-labelled unspecific protein in the tumours of treated mice. These findings indicate that compared with full-length rhCNB, DC protein not only accumulates more effectively in tumour tissues but also persists longer and exhibits more concentrated targeting, underscoring its superior tumour-targeting ability. 3.4. Immunostimulatory effects of the DC protein To evaluate the immunostimulatory effects of the DC protein, immature dendritic cells (imDCs) were treated with PBS (Vehicle), 100 µg/ml DC protein, or 100 µg/ml rhCNB. Following treatment, the cells were collected for analysis of the costimulatory molecules CD80 and CD86, and the culture supernatants were harvested to measure the secretion of the cytokines IL-12 and TNF-α, as well as the chemokine RANTES (CCL5). As shown in Fig. 5 , compared with the blank control, both the DC and rhCNB treatments significantly increased the expression of CD80 (P < 0.0001; Fig. 5 A) and CD86 (P < 0.01; Fig. 5 B), indicating that the truncated variant DC effectively promoted dendritic cell differentiation and maturation. Notably, at 100 µg/ml, compared with rhCNB, the DC protein induced marginally lower CD80 expression (by approximately 9%), although this difference was not statistically significant (P = 0.2357; Fig. 5 A). In contrast, compared with DC, rhCNB induced significantly higher CD86 expression (P = 0.0017; Fig. 5 B). Furthermore, DC effectively stimulated the secretion of TNF-α, IL-12, and RANTES by dendritic cells (Fig. 5 C–E). However, the level of RANTES secretion induced by DC was marginally lower than that induced by rhCNB (by approximately 4.5%), but the difference was not statistically significant (P = 0.0527; Fig. 5 C). Similarly, the DC-induced TNF-α secretion was slightly lower than that induced by rhCNB (by approximately 10%), and this difference was significant (P = 0.0144; Fig. 5 D). Moreover, compared with that in the rhCNB group, IL-12 production in the DC group was substantially lower (by more than 50%, P = 0.0024; Fig. 5 E). Given that TNF-α and IL-12 are critical antitumour cytokines that suppress tumour growth through multiple mechanisms and that RANTES recruits various immune cells to enhance antitumour immunity, these findings collectively demonstrate that the truncated variant DC, like rhCNB, enhances antitumour immune responses by promoting DC maturation and cytokine secretion. The reduced efficacy of DC relative to that of full-length rhCNB may be attributed to structural constraints in protein folding. Nevertheless, the advantages of DC, including its smaller size, targeted delivery capability, and immunostimulatory properties, support its superior potential for future drug development. 3.5 Treatment of H22 tumour-bearing mice with the DC protein We evaluated the antitumour efficacy of DC in H22-transplanted tumour mice, and with increasing length of administration, the tumour volumes in the groups administered 5 mg/kg rhCNB and 5 mg/kg DC were significantly lower compared with those administered normal saline injection (rhCNB group: P < 0.0001; DC group: P = 0.001). No significant difference in efficacy was observed between the DC and rhCNB groups (P = 0.1637; Fig. 6A). At the end of the treatment period, the tumours were excised and weighed. The tumour inhibition rates were 40.5% for the DC group and 53.73% for the rhCNB group, with no statistically significant difference between them (P = 0.2059; Fig. 6B). Compared with the DC group, the rhCNB group exhibited stronger antitumour effects, which is consistent with the enhanced immunostimulatory activity observed in vitro. Notably, the H22 tumour model was established in ICR wild-type mice, in which inherent individual variability in immune function may have contributed to inconsistent treatment responses among the groups. These in vivo results demonstrate that DC retains a substantial portion of the antitumour activity of full-length rhCNB. However, the truncation likely leads to a reduction in its overall antitumour potency. 4. Discussion rhCNB is a genetically engineered antitumour drug under development that exhibits immunostimulatory, antitumour, rapid internalization into diverse tumour cells, and tumour-targeting functions [ 4 – 7 , 10 ]. The C-terminal domain primarily mediates the internalization and targeting of rhCNB [ 9 ]; however, its immunostimulatory antitumour domain remains uncharacterized. Here, we constructed the C-terminal fragment DC (aa 85–169) of rhCNB and demonstrated that this fragment is the critical functional region for the targeting and antitumour activity of rhCNB. CNB is composed of two global domains: a C-terminal domain (DC, 85–169) and an N-terminal domain (DN, aa 1–84), each containing two Ca [2+] binding sites. Herein, multiple truncated variants of rhCNB were engineered, among which DC is a conservatively truncated form [ 9 , 11 ]. This study demonstrated that DC retains the key functional properties of the cellular uptake and in vivo targeting capabilities of rhCNB. However, compared with full-length rhCNB, its immunomodulatory functions were compromised in certain aspects, leading to diminished antitumour efficacy in vivo. These results indicate that the N-terminal EF-hand structures are critical for the proper folding of rhCNB, enabling it to bind more tightly to its receptor complexes. Thus, the N-terminal domains are essential for all the biological functions of rhCNB. CNB is a highly hydrophobic protein, and its surface hydrophobicity is very important for activating CNA phosphatase activity. DC retains a substantially hydrophobic structure and harbours multiple hydrophobic residues in its secondary structure [ 12 , 13 ]. In addition, its hydrophobic characteristics facilitate its purification. In a previous study, we confirmed that rhCNB is an endogenous ligand of TLR4 [ 14 ]. TLR4 recognizes diverse substances, including danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) [ 15 , 16 ]. We speculated that the hydrophobic architecture of DC may align with those of TLR4-recognized DAMPs. This speculation needs further investigation. In addition, among the four EF-hand motifs of rhCNB, the C-terminal EF-hands exhibit higher Ca² binding affinity than their N-terminal counterparts do [ 17 , 18 ], and the crystal structure shows that the formation of the hydrophobic groove by residues 118–123 of CnB is necessary for its interactions with two different immunosuppressant–immunophilin complexes and with CnA [ 19 ]. Like full-length rhCNB, DC encompasses these C-terminal EF-hands, potentially enabling its antitumour, cellular uptake and tumour targeting abilities. Peptide‒drug conjugates (PDCs) are structured similarly to ADCs; however, PDCs possess several distinct advantages over ADCs. Their small size promotes deep penetration into tumour tissues and uptake by cancer cells. Additionally, the PDC platform is associated with lower immunogenicity and leverages the biological activities of peptides to improve treatment outcomes. The DC protein, characterized by its low molecular weight of approximately 10 kDa, can target tumour tissues and be internalized by tumour cells. This functional profile defines its utility in the design of peptide–drug conjugates (PDCs), positioning it as a highly promising platform for novel cancer therapeutics [ 20 , 21 , 22 ]. The application of nanoparticles modified with targeting ligands to deliver chemotherapeutic agents directly to tumour sites is a promising strategy for cancer therapy. The delivery of chemotherapeutic drugs can effectively improve their therapeutic effects and reduce their toxicity [ 23 , 24 , 25 ]. DC exhibits excellent properties for targeting tumour tissues and internalization into tumour cells. These characteristics enable its use in the construction of multifunctional nanoparticles and establish them as ideal targeting moieties for chemotherapeutic delivery systems. 5. Conclusions In conclusion, this study successfully constructed and characterized a truncated DC variant (aa 85–169) of rhCNB as the critical functional domain mediating its antitumour activity. Our experimental results demonstrate that DC retains the key biological functions exhibited by the intact rhCNB protein, including its immunomodulatory effects, tumour-targeting capability and internalization function of tumour cells. These findings not only provide crucial insights into the structure‒function relationship of rhCNB at the molecular level but also establish DC as a promising therapeutic candidate with significant potential for clinical development. Future investigations should focus on further optimizing this domain for enhanced therapeutic efficacy and exploring its clinical applications, either as a monotherapy or in rational combination regimens with existing anticancer agents. Abbreviations rhCNB recombinant human Calcineurin B subunit PTX paclitaxel BMDCs Bone marrow-derived dendritic cells Declarations Funding: This research was funded by the National Natural Science Foundation of China ( 82172627). Ethics statement ： All animal experiments were conducted in 2020 in accordance with the institutional guidelines for the care and use of laboratory animals issued by the China Public Health Service. The study protocol was reviewed and approved by the Animal Ethics Committee of Beijing Normal University (Approval No. CLS-EAW-2013-015) and remained valid for the experimental procedures performed in this study. According to the approved protocol, the maximal tumor size permitted by the ethics committee was 3,000 mm³or 10% of the body weight (the average body weight of ICR mice after treatment was approximately 40 g). Throughout the study, the tumor burden in all mice remained below these limits, and no animals exhibited ulceration, necrosis, or signs of distress. All mice were housed in a specific pathogen-free (SPF) facility under controlled temperature and a 12-hour light/dark cycle, with ad libitum access to food and water. All efforts were made to minimize animal suffering and to reduce the number of animals used. Consent to Publish : All authors have read and approved the final version of the manuscript and consent to its publication. Consent to participate : Not applicable. Author Contributions: Study design was carried out by Jinju Yang and Qun Wei, and the experiments was performed by Ziwei Zhu,.The manuscript was written by Jinju Yang and Ziwei Zhu, All authors have read and agreed to the published version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Eur J Med Chem. 2024. 5:265:116119. Dongyuan Wang F, Yin Z, Li Y, Zhang C. Current progress and remaining challenges of peptide-drug conjugates (PDCs): next generation of antibody-drug conjugates (ADCs)? J Nanobiotechnol. 2025;23(1):305. 10.1186/s12951-025-03277-2 . Vahab Alamdari-Palangi,. Khojaste Rahimi Jaberi, etc. Recent advances and applications of peptide-agent conjugates for targeting tumor cells. J Cancer Res Clin Oncol. 2023;;149(16):15249–73. Zhuxuan G. ,uan, etc. Peptide ligand-mediated targeted drug delivery of nanomedicines. Biomater Sci. 2019; 7(2): 461–471. Wang C, Zhang S. Advantages of nanomedicine in cancer therapy: a review. ACS Appl Nano Mater. 2023);;6(24):22594–610. https://doi:10.1021/acsanm.3c04487 . (. Manzari MT, Shamay Y, Kiguchi H, Rosen N, Scaltriti M, Heller DA. Targeted drug delivery strategies for precision medicines. Nat Rev Mater. 2021);;6(4):351–70. https://doi:10.1038/s41578-020-00269-6 . (. Additional Declarations No competing interests reported. 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15:34:14","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83808,"visible":true,"origin":"","legend":"","description":"","filename":"353fe1f9935d415197355a30454b54411structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/8c3223bb2745f1c9f4b8871b.xml"},{"id":97714533,"identity":"9f778e26-8836-48a8-8c9b-e968d59b8f1b","added_by":"auto","created_at":"2025-12-08 14:24:25","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91419,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/d8687554d921987f2ba3ed21.html"},{"id":97895399,"identity":"2c6a2ab5-83e5-488f-9ec7-64d79f3977df","added_by":"auto","created_at":"2025-12-10 15:34:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":140795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction, expression and purification of the truncated variant DC based on rhCNB. \u003c/strong\u003e(a). 3D structures of the truncated variant DC (green, right panel) generated with PyMOL software. (b). Schematic diagram of the rhCNB and the variant DC. Full-length rhCNB contains four EF-hand motifs (1–4), whereas the DC variant comprises only the two C-terminal EF-hands (3 and 4). (c, d). SDS‒PAGE and Western blot analysis of DC expression in IPTG-induced clones. The DC-positive clones were induced for DC expression, and the bacterial pellets were collected and analysed by SDS‒PAGE. Lane 1, protein marker (170, 130, 95, 72, 55, 43, 34, 26, 17, and 10 kDa); Lane 2, lysate from the negative control; Lanes 3–8, lysates from DC-positive clones. (e). Chromatogram of the multistep purification process for the DC protein. The clarified supernatant was subjected to phenyl hydrophobic interactions, Sephadex G-25 buffer exchange, and DEAE ion-exchange chromatography. (f). SDS‒PAGE analysis of samples from the DC purification process. Lane 1, protein marker; Lane 2, insoluble fraction after sonication; Lane 3, soluble supernatant after sonication; Lane 4, soluble fraction after heat treatment; Lane 5, sample loaded onto a phenyl column; Lane 6, phenyl flow-through; Lane 7, phenyl wash; Lane 8, phenyl elution; Lane 9, sample after Sephadex G-25 buffer exchange; Lane 10, target fraction from Sephadex G-25; Lane 11, sample loaded onto a DEAE column; Lane 12, DEAE elution fraction. (g). Western blot analysis of the final purified DC product. The final DC product was separated and blotted with an anti-rhCNB polyclonal antibody. (h). Determination of the enzymatic activity of the final DC products by the pNDD method. The reaction mixture, containing the DC product, CNA, rhCNB and the substrate pNDD, was incubated, terminated, and measured at 410 nm. (n=3, Data are the means ± SE of 3 independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/77b4f9858b4cc0b5ec0cfa92.png"},{"id":97895473,"identity":"425fcd43-7e65-41b7-86db-cf9fdefa521a","added_by":"auto","created_at":"2025-12-10 15:34:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe DC protein specifically enters tumour cells. \u003c/strong\u003eSMMC-7721 cells were incubated with\u003cstrong\u003e \u003c/strong\u003e5 μM 5-FAM labelled DC protein, 5-FAM labelled rhCNB protein or unlabelled DC protein, and images were captured with a Zeiss LSM700 confocal laser scanning microscope (a).\u003cstrong\u003e \u003c/strong\u003eDifferent tumour cells were seeded into 96-well plates and treated with various concentrations of vehicle, rhCNB, DC, PTX, or combinations of DC/PTX and rhCNB/PTX. Cell proliferation was determined by the modified MTT method. The DC and rhCNB proteins significantly potentiated PTX cytotoxicity against SMMC-7721 tumour cells (b), MHCC-97H tumour cells (c), HepG2 tumour cells (d), and CT26 tumour cells (e). (n=3, Data are the means ± SE of 3 independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/9f38d9dca9bf3860535638e6.png"},{"id":97893699,"identity":"a457c1c0-1d99-48e6-a673-bcd06049f73a","added_by":"auto","created_at":"2025-12-10 15:30:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":337465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the tumour-targeting ability of DC in SMMC-7721 tumour-bearing nude mice and H22 tumour-bearing ICR mice. \u003c/strong\u003e(a, c). In vivo real-time NIRF images of Cy7-labelled rhCNB and DC at various time points post injection. (b, d). Ex vivo NIRF images and mean fluorescence intensities of Cy7-labelled rhCNB and DC at various time points postinjection. Cy7-labelled rhCNB or Cy7-labelled DC was injected into tumour-bearing nude mice via the tail vein in vivo. NIRF images were captured at 6, 8, 10, 24 and 36 hours after injection. The mice were euthanized at 6, 24 and 36 hours post-injection for tumour resection and imaging analysis in ImageJ software (n=3; **P\u0026lt;0.01; *P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/93c412b922576c7ed242f0c3.png"},{"id":97714523,"identity":"ae5c89e0-d7b3-4e0e-b9ea-866fbc1c8525","added_by":"auto","created_at":"2025-12-08 14:24:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunostimulatory effects of the DC protein.\u003c/strong\u003e (a, b). The DC protein upregulated the expression of the costimulatory molecules CD80 and CD86 on the surface of BMDCs. (c–e). The DC protein promoted the secretion and production of antitumour cytokines. BMDCs were treated with rhCNBor DCfor 48 h, and the cells and the supernatants were collected for FCM analysis and determination of cytokines and chemokines (n=3; data are presented as the means ± SEs of three independent experiments. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/8ab0fa6a3db245251016279d.png"},{"id":97894149,"identity":"2e251185-2779-42df-86fa-a3350db958f3","added_by":"auto","created_at":"2025-12-10 15:31:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42893,"visible":true,"origin":"","legend":"\u003cp\u003eThe antitumour effects of rhCNB and its truncated variant DC on H22 tumours. H22 cells were inoculated into ICR mice, and the mice received daily intraperitoneal injections of saline, 5 mg/kg rhCNB or 5 mg/kg DC. Tumour volumes were measured every 2 days (a). After 25 days, the tumours were removed and weighed (b). (Data are the means ± SE. n = 6–8; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P\u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/ccd9e6e717ee0e624d046f74.png"},{"id":101852266,"identity":"8a81e9ce-532b-43fe-a915-28990b4880f0","added_by":"auto","created_at":"2026-02-04 10:11:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1837039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/ea15e4fa-cb2b-41e0-a7b0-8c5e1bd783df.pdf"},{"id":97895645,"identity":"cd99586c-6110-4baa-a030-1e9b74885db3","added_by":"auto","created_at":"2025-12-10 15:34:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3734975,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7943596/v1/c796743bb1fefb1bc42ae75f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional identification of the C-terminal domain of rhCNB","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe calmodulin phosphatase B subunit (CNB) is the regulatory subunit of calmodulin phosphatase (CN), and its main function in vivo is to regulate the activity of calmodulin phosphatase A subunit (CNA) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies have indicated that CNB has other physiological functions independent of CNA; for example, CNB deficiency is associated with a high risk of squamous cell carcinoma [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eResearch in our laboratory revealed that recombinant CNB significantly inhibited tumour growth and prolonged survival in various mouse models [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Further studies indicated that it promoted dendritic cell maturation and antigen presentation, increased the phagocytic activity of macrophages and natural killer (NK) cells, and triggered the secretion of proinflammatory cytokines and chemokines. These properties enable its use as both a tumour vaccine adjuvant and an immunomodulator, effectively inducing cellular and humoral immunity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMammalian CNB is composed of 169 amino acids and has 4 EF-hand structures, belonging to the calcium-binding protein family. The typical structural feature of the EF hand is a spiral structure, and each EF hand structure can bind to a calcium ion. Different EF-hand regions exhibit vastly different binding affinities for calcium ions, varying by up to a million-fold [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The main segments mediating the internalization and targeting of rhCNB are located at the C-terminus [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; however, it is not known if the main segment mediating the antitumour and immune activation functions is concentrated at the C-terminus. To explore the main antitumour functional segments of rhCNB, we prepared a C-terminal fragment of rhCNB (amino acids 85\u0026ndash;169) and characterized its function. The results indicated that the C-terminus of rhCNB is the key domain responsible for antitumour activity.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell lines and animal culture\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe cell lines used in this study included the human hepatocellular carcinoma cell lines SMMC-7721, HepG2, and MHCC-97H; the mouse hepatocellular carcinoma cell line H22; and the mouse colon carcinoma cell line CT26. SMMC-7721, HepG2, H22, and CT26 cells were purchased from ATCC, and MHCC-97H cells were obtained from the Shanghai Institute of Cell Biology. These cell lines were confirmed to be free of Mycoplasma contamination through PCR and fluorescence staining (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFemale ICR (8 weeks old, weighing 30\u0026ndash;40 g) and BALB/c nude mice (6\u0026ndash;8 weeks old, weighing 20\u0026ndash;22 g) were used in this study. These mice were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Construction, Expression and Purification of DC\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe DC fragment was obtained from the rhCNB (P63098) plasmid and constructed by restriction enzyme digestion and ligation. The plasmids containing the correct inserts were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) competent cells for protein expression. Seven individual transformants were induced by IPTG for high-expression strain screening, and the expression products were identified by SDS‒PAGE and Western blot analysis. The selected high-expression strain was expanded for large-scale protein production.\u003c/p\u003e\u003cp\u003eThe purification of DC was performed using the method previously established for rhCNB. Briefly, the bacterial cells were harvested and resuspended in Buffer A (pH 7.4, 20 mM Tris-HCl). The suspension was subsequently sonicated, boiled in a water bath for 20 minutes, and centrifuged at 10,000 rpm for 20 minutes to collect the supernatant. The supernatant containing recombinant DC protein was loaded on phenyl hydrophobic chromatography equilibrated with Buffer B (20 mM Tris-HCl, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, and 0.5 M NaCl, pH 7.4), and the DC protein was subsequently eluted with Buffer C (20 mM Tris-HCl, 0.5 mM EGTA, pH 7.4). The eluted fraction from the phenyl column was lyophilized and subjected to buffer exchange into 20 mM phosphate buffer (PB, pH 7.4) using a Sephadex G25 desalting column. The collected fraction was further purified by DEAE ion-exchange chromatography, which was primarily employed for endotoxin removal. The sample was loaded in Buffer D (20 mM PB, pH 7.4) and eluted with Buffer E (20 mM PB, 100 mM NaCl, pH 7.4). Finally, the purified fraction was concentrated by freeze-drying. The concentration of the final product was measured by a BCA assay, the endotoxin content was detected using limulus reagent, the purity was analysed by SDS‒PAGE, and the activity was determined by the pNPP method.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.4. DC activity determination\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe activity of the purified DC was determined by the pNPP (p-nitrophenyl phosphate) method, a simple, widely used colorimetric assay for measuring phosphatase activity. Briefly, enzyme dilutions or diluted CNA were separately added to their corresponding tubes and precooled on ice for 5 min. Subsequently, the activity assay solution, either alone or with purified DC or rhCNB, was introduced, and the mixtures were incubated at 30\u0026deg;C for 20 min. The reactions were stopped, and the absorbance at 410 nm (OD₄₁₀) was recorded. Each treatment was performed with three technical replicates, and the entire experiment was independently repeated three times. In this setup, CNA alone served as the negative control, CNA\u0026thinsp;+\u0026thinsp;rhCNB as the positive control, enzyme dilution alone as the blank control, and CNA combined with DC (CNA\u0026thinsp;+\u0026thinsp;DC) constituted the test group.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Cellular Uptake of Exogenous DC\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e5-FAM-labelled rhCNB and DC were prepared for cellular uptake analysis. Briefly, an equimolar amount of 5-FAM was mixed with either DC or rhCNB and incubated in the dark at room temperature for 1 h. The mixture was dialyzed overnight at 4\u0026deg;C against PBS using a 3.5 kDa cut-off dialysis membrane to remove unbound dye. Cells (2 \u0026times; 10⁵) seeded in 60 mm Petri dishes were treated with 5 \u0026micro;M 5-FAM-labelled rhCNB, 5-FAM-labelled DC, or unlabelled DC (negative control) and incubated at 37\u0026deg;C for 30 minutes. The cells were subsequently washed three times with PBS, exposed to acid-stripping buffer (pH 5.0 Gly-HCl buffer), and fixed with 4% paraformaldehyde. Cellular uptake was visualized using a Zeiss confocal fluorescence microscope, and fluorescence intensity was quantified with ImageJ software by measuring the mean grey value of the images.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.6. In Vivo Imaging\u003c/h2\u003e\u003cp\u003eDC or rhCNB was labelled with Cy7-SE following the procedure described in Section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.5\u003c/span\u003e. SMMC-7721 tumour-bearing mice or H22 tumour-bearing mice were established by subcutaneously injecting 1\u0026times;10⁶ cells into the right armpit. When the tumour volume reached approximately 100 mm\u0026sup3;, the mice were randomly assigned to one of three groups (n\u0026thinsp;=\u0026thinsp;9). Each group received a tail vein injection of 100 \u0026micro;g of Cy7-labelled DC, Cy7-labelled rhCNB, or unlabeled DC (as a blank control). In vivo imaging was performed at 6, 8, 10, 24, and 36 h post-injection using a Revvity IVIS Spectrum system. During imaging, mice were anesthetized with isoflurane (2\u0026ndash;3% for induction and 1\u0026ndash;2% for maintenance) in oxygen. At 6, 24, and 36 h post-injection, the mice were deeply anesthetized with isoflurane followed by cervical dislocation for euthanasia. The tumors and major organs (heart, liver, spleen, lung, and kidney) were collected for ex vivo fluorescence imaging. The fluorescence intensity of the excised tumors was quantified using ImageJ software by measuring the mean RGB values of the images.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Isolation and Induction of Mouse Bone Marrow-Derived Dendritic Cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBone marrow cells were isolated from ICR mice by flushing the bone marrow cavity with RPMI-1640 medium. The cell suspension was gently dispersed, filtered through a 200-mesh nylon sieve, and centrifuged at 1500 \u0026times; g for 5 min. After two washes with PBS, the cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 25 ng/mL recombinant mGM-CSF and 25 ng/mL mIL-4. The medium was replaced every two days, and CD11c staining was performed every three days to monitor differentiation. On Day 6, nonadherent and loosely adherent cells were collected as immature dendritic cells (imDCs). For stimulation, imDCs were seeded in 12-well plates at 1\u0026times;10⁶ cells/well. The following day, the cells were treated with 100 \u0026micro;g/mL rhCNB, 100 \u0026micro;g/mL DC, or PBS (as a vehicle control) and coincubated for 48 h. The expression of the surface costimulatory molecules CD80 and CD86 was subsequently analysed by flow cytometry (FCM), and the levels of cytokines and chemokines in the supernatants were determined using commercial ELISA kits.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.8. MTT Assay for Cell Proliferation\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMHCC-97H, HepG2, CT26, and SMMC-7721 cells were seeded into 96-well plates at 4000 cells/well. On the following day, the culture medium was replaced with fresh medium containing either rhCNB or DC (at concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.156, or 0 \u0026micro;M), PTX (at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, or 0 \u0026micro;M), or combinations of DC and PTX or rhCNB and PTX. The combination treatments consisted of fixed ratios of rhCNB (or DC) and PTX (5 \u0026micro;M\u0026thinsp;+\u0026thinsp;1 \u0026micro;M), (2.5 \u0026micro;M\u0026thinsp;+\u0026thinsp;0.5 \u0026micro;M), (1.25 \u0026micro;M\u0026thinsp;+\u0026thinsp;0.25 \u0026micro;M), (0.625 \u0026micro;M\u0026thinsp;+\u0026thinsp;0.0625 \u0026micro;M), (0.3125 \u0026micro;M\u0026thinsp;+\u0026thinsp;0.03125 \u0026micro;M), (0.156 \u0026micro;M\u0026thinsp;+\u0026thinsp;0.0156 \u0026micro;M), and (0 \u0026micro;M\u0026thinsp;+\u0026thinsp;0 \u0026micro;M). After the compounds were added, the cells were incubated for 48 h. Cell proliferation was assessed using a modified MTT assay.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Tumour Treatment\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eICR mice (6\u0026ndash;8 weeks old, body weight 30\u0026ndash;40 g) were randomly divided into 3 groups (n\u0026thinsp;=\u0026thinsp;10) and received daily intraperitoneal injections (0.2 mL) of normal saline, 5 mg/kg rhCNB, or 5 mg/kg DC. After three administrations, 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e H22 cells were transplanted into the right armpits of ICR mice. Tumour volumes were measured using Vernier callipers every 2 days beginning on Day 5 after tumour challenge. After 25 days, Mice were deeply anesthetized with isoflurane followed by cervical dislocation, and the tumours were removed and weighed.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test in GraphPad Prism 6. A P value of \u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance, and the significance levels are denoted as follows: *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.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Construction and Purification of the Truncated DC Protein\u003c/h2\u003e\u003cp\u003eWe engineered a truncated variant of rhCNB, designated DC, which consists of the C-terminal region (aa 85\u0026ndash;169) and corresponds to half of the full-length rhCNB sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, green). RhCNB contains four EF-hand motifs; however, the DC variant retains only the two C-terminal motifs (EF-hands 3 and 4), which are critical for its function (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). SDS‒PAGE and Western blot analyses of IPTG-induced DC transformant colonies confirmed the successful expression of the DC protein. The recombinant protein had the expected molecular weight of approximately 10 kDa and was specifically recognized by an anti-rhCNB polyclonal antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Clone #4 (marked by a white box in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), which exhibited optimal expression characteristics, was selected as the production strain.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe selected production strain (Clone #4) was subjected to large-scale cultivation. Following cell lysis via sonication, the DC protein was detected primarily in the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Lanes 2\u0026ndash;3). A subsequent heat treatment step effectively enriched the DC protein in the boiled supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Lane 4). This clarified supernatant was then purified through a sequential process of phenyl hydrophobic chromatography, Sephadex G25 buffer exchange, and DEAE ion-exchange chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Analysis of the eluted fractions by SDS\u0026ndash;PAGE demonstrated that the phenyl chromatography step significantly increased the purity of the target protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Lanes 5\u0026ndash;8). Buffer exchange using a Sephadex G25 column maintained this high purity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Lanes 9\u0026ndash;10). Finally, the DEAE chromatography step yielded a highly pure final product that met the required endotoxin specification of less than 0.1 EU/mg (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Lanes 11\u0026ndash;12).\u003c/p\u003e\u003cp\u003eWestern blot analysis confirmed that the purified protein was the target DC variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). CNB, as the regulatory subunit of calcineurin, regulates the activity of the catalytic subunit CNA. To functionally characterize the DC protein, we utilized a pNDD assay to determine whether the DC protein could enhance the catalytic activity of CNA. The results indicated a significant increase in CNA activity by the DC protein (P\u0026thinsp;=\u0026thinsp;0.0085; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These findings verified the successful preparation of the target DC protein, which not only met purity specifications but also retained the intended biological function.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. DC Rapidly Enters Tumour Cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOur previous studies demonstrated that rhCNB can be rapidly internalized into tumour cells. The truncated variant Trun3 (aa 124\u0026ndash;169), which was identified as the main segment for rhCNB internalization, can also quickly enter tumour cells and is only slightly weaker than full-length rhCNB. The DC fragment (aa 85\u0026ndash;169), which encompasses the complete Trun3 domain, was hypothesized to retain comparable internalization capacity. To detect the effects of DC internalization, labelled DC, rhCNB and unlabelled DC were added to SMMC-7721 cells, and the results of confocal imaging revealed that DC and rhCNB rapidly entered the cells within 15 minutes and that the fluorescence intensities of DC and rhCNB did not significantly differ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Fig. S2).\u003c/p\u003e\u003cp\u003eThe tumour-targeting and tumour-internalizing abilities of therapeutic proteins are critical for achieving targeted delivery of chemotherapeutic drugs. Such targeted delivery enables chemotherapeutic agents to be transported directly into tumour sites, effectively killing cancer cells while minimizing toxicity to normal organs and tissues, thereby substantially improving therapeutic outcomes. To assess whether DC or rhCNB enhances the cytotoxic effect of chemotherapeutic agents on tumour cells via internalization, we evaluated their combined effects with paclitaxel (PTX) across several tumour cell models. The results revealed that compared with vehicle alone, PTX alone, as well as in combination with DC/PTX and rhCNB/PTX, significantly suppressed tumour cell growth. At higher concentrations, no significant differences were observed among the three treatment groups. However, at lower concentrations, both combination regimens resulted in markedly stronger inhibition than PTX alone did (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D, E), whereas no notable difference was detected between the two combination groups. Treatment with rhCNB or DC alone did not inhibit the growth of any of the tumour cell lines tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Further data indicated that both DC and rhCNB significantly increased the cytotoxicity of PTX in the human hepatocellular carcinoma cell lines SMMC-7721 (rhCNB/PTX: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; DC/PTX: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; PTX alone: P\u0026thinsp;=\u0026thinsp;0.0008; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and MHCC-97H (rhCNB/PTX: P\u0026thinsp;=\u0026thinsp;0.0038; DC/PTX: P\u0026thinsp;=\u0026thinsp;0.0439; PTX alone: P\u0026thinsp;=\u0026thinsp;0.0692; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC); HepG2 cells (rhCNB/PTX: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; DC/PTX: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; PTX alone: P\u0026thinsp;=\u0026thinsp;0.0034; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), as well as in the mouse colon carcinoma cell line CT26 (rhCNB/PTX: P\u0026thinsp;=\u0026thinsp;0.0038; DC/PTX: P\u0026thinsp;=\u0026thinsp;0.0055; PTX alone: P\u0026thinsp;=\u0026thinsp;0.0692; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In summary, these findings suggest that both DC and rhCNB facilitate PTX encapsulation and internalization through their tumour-homing ability, thereby promoting tumour cell apoptosis, lowering the effective dose of PTX needed, and improving its safety profile.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. The DC Protein Also Targets Tumours\u003c/h2\u003e\u003cp\u003ePrevious studies have demonstrated that both exogenous rhCNB and its truncated variant Trun3 exhibit tumour-targeting properties in vivo. In this study, we evaluated the targeting efficacy of DC in SMMC-7721 tumour-bearing BALB/c nude mice and H22 tumour-bearing ICR mice.\u003c/p\u003e\u003cp\u003eIn SMMC-7721 tumour-bearing mice, both Cy7-labelled DC and Cy7-labelled rhCNB were rapidly distributed and began to accumulate in tumour tissue within 6 hours post-injection, and the signals persisted for up to 24 hours. Notably, fluorescence remained detectable at the tumour site in the Cy7-labelled DC group at the 36-hour time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Ex vivo fluorescence imaging of the resected tumours further confirmed the retention of both agents within the tumour tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Quantitative analysis of tumour fluorescence revealed that compared with the rhCNB group, the DC group exhibited a slightly greater signal intensity at 6 hours, but the difference was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). By 24 and 36 hours, the fluorescence intensity in the DC group was markedly greater than that in the rhCNB group, and this difference became statistically significant (24 h: P\u0026thinsp;=\u0026thinsp;0.0116. 36 h: P\u0026thinsp;=\u0026thinsp;0.0296. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eIn H22 tumour-bearing mice, both labelled proteins markedly accumulated and persisted within tumour tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). At the 6-hour time point, the rhCNB group demonstrated significantly greater fluorescence intensity than the DC group did (P\u0026thinsp;=\u0026thinsp;0.0359. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, this trend was reversed by 24 and 36 hours, at which time the fluorescence of DC was significantly stronger than that of rhCNB (24 h: P\u0026thinsp;=\u0026thinsp;0.0149. 36 h: P\u0026thinsp;=\u0026thinsp;0.0404. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eIn this study, unlabelled DC served as a blank control. We did not establish a free Cy7 group or a Cy7-labelled unspecific protein group because in a previous study, we detected no significant accumulation of free Cy7 or Cy7-labelled unspecific protein in the tumours of treated mice. These findings indicate that compared with full-length rhCNB, DC protein not only accumulates more effectively in tumour tissues but also persists longer and exhibits more concentrated targeting, underscoring its superior tumour-targeting ability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Immunostimulatory effects of the DC protein\u003c/h2\u003e\u003cp\u003eTo evaluate the immunostimulatory effects of the DC protein, immature dendritic cells (imDCs) were treated with PBS (Vehicle), 100 \u0026micro;g/ml DC protein, or 100 \u0026micro;g/ml rhCNB. Following treatment, the cells were collected for analysis of the costimulatory molecules CD80 and CD86, and the culture supernatants were harvested to measure the secretion of the cytokines IL-12 and TNF-α, as well as the chemokine RANTES (CCL5). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, compared with the blank control, both the DC and rhCNB treatments significantly increased the expression of CD80 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and CD86 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that the truncated variant DC effectively promoted dendritic cell differentiation and maturation. Notably, at 100 \u0026micro;g/ml, compared with rhCNB, the DC protein induced marginally lower CD80 expression (by approximately 9%), although this difference was not statistically significant (P\u0026thinsp;=\u0026thinsp;0.2357; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, compared with DC, rhCNB induced significantly higher CD86 expression (P\u0026thinsp;=\u0026thinsp;0.0017; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, DC effectively stimulated the secretion of TNF-α, IL-12, and RANTES by dendritic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;E). However, the level of RANTES secretion induced by DC was marginally lower than that induced by rhCNB (by approximately 4.5%), but the difference was not statistically significant (P\u0026thinsp;=\u0026thinsp;0.0527; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Similarly, the DC-induced TNF-α secretion was slightly lower than that induced by rhCNB (by approximately 10%), and this difference was significant (P\u0026thinsp;=\u0026thinsp;0.0144; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Moreover, compared with that in the rhCNB group, IL-12 production in the DC group was substantially lower (by more than 50%, P\u0026thinsp;=\u0026thinsp;0.0024; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Given that TNF-α and IL-12 are critical antitumour cytokines that suppress tumour growth through multiple mechanisms and that RANTES recruits various immune cells to enhance antitumour immunity, these findings collectively demonstrate that the truncated variant DC, like rhCNB, enhances antitumour immune responses by promoting DC maturation and cytokine secretion. The reduced efficacy of DC relative to that of full-length rhCNB may be attributed to structural constraints in protein folding. Nevertheless, the advantages of DC, including its smaller size, targeted delivery capability, and immunostimulatory properties, support its superior potential for future drug development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Treatment of H22 tumour-bearing mice with the DC protein\u003c/h2\u003e\u003cp\u003eWe evaluated the antitumour efficacy of DC in H22-transplanted tumour mice, and with increasing length of administration, the tumour volumes in the groups administered 5 mg/kg rhCNB and 5 mg/kg DC were significantly lower compared with those administered normal saline injection (rhCNB group: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; DC group: P\u0026thinsp;=\u0026thinsp;0.001). No significant difference in efficacy was observed between the DC and rhCNB groups (P\u0026thinsp;=\u0026thinsp;0.1637; Fig.\u0026nbsp;6A). At the end of the treatment period, the tumours were excised and weighed. The tumour inhibition rates were 40.5% for the DC group and 53.73% for the rhCNB group, with no statistically significant difference between them (P\u0026thinsp;=\u0026thinsp;0.2059; Fig.\u0026nbsp;6B). Compared with the DC group, the rhCNB group exhibited stronger antitumour effects, which is consistent with the enhanced immunostimulatory activity observed in vitro. Notably, the H22 tumour model was established in ICR wild-type mice, in which inherent individual variability in immune function may have contributed to inconsistent treatment responses among the groups. These in vivo results demonstrate that DC retains a substantial portion of the antitumour activity of full-length rhCNB. However, the truncation likely leads to a reduction in its overall antitumour potency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003erhCNB is a genetically engineered antitumour drug under development that exhibits immunostimulatory, antitumour, rapid internalization into diverse tumour cells, and tumour-targeting functions [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The C-terminal domain primarily mediates the internalization and targeting of rhCNB [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; however, its immunostimulatory antitumour domain remains uncharacterized. Here, we constructed the C-terminal fragment DC (aa 85\u0026ndash;169) of rhCNB and demonstrated that this fragment is the critical functional region for the targeting and antitumour activity of rhCNB.\u003c/p\u003e\u003cp\u003eCNB is composed of two global domains: a C-terminal domain (DC, 85\u0026ndash;169) and an N-terminal domain (DN, aa 1\u0026ndash;84), each containing two Ca\u003csup\u003e[2+]\u003c/sup\u003e binding sites. Herein, multiple truncated variants of rhCNB were engineered, among which DC is a conservatively truncated form [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This study demonstrated that DC retains the key functional properties of the cellular uptake and \u003cem\u003ein vivo\u003c/em\u003e targeting capabilities of rhCNB. However, compared with full-length rhCNB, its immunomodulatory functions were compromised in certain aspects, leading to diminished antitumour efficacy in vivo. These results indicate that the N-terminal EF-hand structures are critical for the proper folding of rhCNB, enabling it to bind more tightly to its receptor complexes. Thus, the N-terminal domains are essential for all the biological functions of rhCNB.\u003c/p\u003e\u003cp\u003eCNB is a highly hydrophobic protein, and its surface hydrophobicity is very important for activating CNA phosphatase activity. DC retains a substantially hydrophobic structure and harbours multiple hydrophobic residues in its secondary structure [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, its hydrophobic characteristics facilitate its purification. In a previous study, we confirmed that rhCNB is an endogenous ligand of TLR4 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. TLR4 recognizes diverse substances, including danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We speculated that the hydrophobic architecture of DC may align with those of TLR4-recognized DAMPs. This speculation needs further investigation. In addition, among the four EF-hand motifs of rhCNB, the C-terminal EF-hands exhibit higher Ca\u0026sup2; binding affinity than their N-terminal counterparts do [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and the crystal structure shows that the formation of the hydrophobic groove by residues 118\u0026ndash;123 of CnB is necessary for its interactions with two different immunosuppressant\u0026ndash;immunophilin complexes and with CnA [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Like full-length rhCNB, DC encompasses these C-terminal EF-hands, potentially enabling its antitumour, cellular uptake and tumour targeting abilities.\u003c/p\u003e\u003cp\u003ePeptide‒drug conjugates (PDCs) are structured similarly to ADCs; however, PDCs possess several distinct advantages over ADCs. Their small size promotes deep penetration into tumour tissues and uptake by cancer cells. Additionally, the PDC platform is associated with lower immunogenicity and leverages the biological activities of peptides to improve treatment outcomes. The DC protein, characterized by its low molecular weight of approximately 10 kDa, can target tumour tissues and be internalized by tumour cells. This functional profile defines its utility in the design of peptide\u0026ndash;drug conjugates (PDCs), positioning it as a highly promising platform for novel cancer therapeutics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe application of nanoparticles modified with targeting ligands to deliver chemotherapeutic agents directly to tumour sites is a promising strategy for cancer therapy. The delivery of chemotherapeutic drugs can effectively improve their therapeutic effects and reduce their toxicity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. DC exhibits excellent properties for targeting tumour tissues and internalization into tumour cells. These characteristics enable its use in the construction of multifunctional nanoparticles and establish them as ideal targeting moieties for chemotherapeutic delivery systems.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn conclusion, this study successfully constructed and characterized a truncated DC variant (aa 85\u0026ndash;169) of rhCNB as the critical functional domain mediating its antitumour activity. Our experimental results demonstrate that DC retains the key biological functions exhibited by the intact rhCNB protein, including its immunomodulatory effects, tumour-targeting capability and internalization function of tumour cells. These findings not only provide crucial insights into the structure‒function relationship of rhCNB at the molecular level but also establish DC as a promising therapeutic candidate with significant potential for clinical development. Future investigations should focus on further optimizing this domain for enhanced therapeutic efficacy and exploring its clinical applications, either as a monotherapy or in rational combination regimens with existing anticancer agents.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003erhCNB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 453px;\"\u003e\n \u003cp\u003erecombinant human Calcineurin B subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003ePTX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 453px;\"\u003e\n \u003cp\u003epaclitaxel\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003eBMDCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 453px;\"\u003e\n \u003cp\u003eBone marrow-derived dendritic cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was funded by the National Natural Science Foundation of China ( 82172627).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eAll animal experiments were conducted in 2020 in accordance with the institutional guidelines for the care and use of laboratory animals issued by the China Public Health Service. The study protocol was reviewed and approved by the Animal Ethics Committee of Beijing Normal University (Approval No. CLS-EAW-2013-015) and remained valid for the experimental procedures performed in this study. According to the approved protocol, the maximal tumor size permitted by the ethics committee was 3,000 mm\u0026sup3;or 10% of the body weight (the average body weight of ICR mice after treatment was approximately 40 g). Throughout the study, the tumor burden in all mice remained below these limits, and no animals exhibited ulceration, necrosis, or signs of distress. All mice were housed in a specific pathogen-free (SPF) facility under controlled temperature and a 12-hour light/dark cycle, with ad libitum access to food and water. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eAll authors have read and approved the final version of the manuscript and consent to its publication.\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eStudy design was carried out by Jinju Yang and Qun Wei, and the experiments was performed by Ziwei Zhu,.The manuscript was written by Jinju Yang and Ziwei Zhu, All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eDual Publication\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis manuscript has not been published previously and is not under consideration for publication elsewhere.\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eAll listed authors have made substantial, direct, and intellectual contributions to the work and approved it for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHemenway CS, Heitman J. 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(.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"rhCNB, tumour therapy, immunostimulation, targeting","lastPublishedDoi":"10.21203/rs.3.rs-7943596/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7943596/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecombinant human Calcineurin B subunit (rhCNB) has emerged as a promising antitumour candidate. Its antitumour activity is mediated by the secretion of proinflammatory cytokines and chemokines from both innate and adaptive immune cells, thereby enhancing their antigen-presenting capacity. Moreover, rhCNB is rapidly internalized by various tumour cells, demonstrating specific tumour-targeting properties. Previous studies have revealed that the C-terminal domain is responsible for the internalization and targeting ability of rhCNB; however, the precise antitumour functional domain remains unexplored. To address this, we engineered a truncated variant (designated DC, comprising amino acids 85\u0026ndash;169 of rhCNB). Functional characterization revealed that DC promoted the maturation and differentiation of bone marrow-derived dendritic cells (BMDCs), stimulated the production of cytokines by BMDCs, were internalized into tumour cells, accumulated at tumour sites, and synergistically enhanced the tumoricidal activity of paclitaxel. Administration of the same dose (5 mg/kg) of DC and rhCNB to H22 tumour-bearing mice resulted in tumour growth inhibition rates of 40.5% and 53.73%, respectively, with no statistically significant difference between the two treatments. Collectively, these findings identify DC as the core functional domain responsible for the antitumour effects of rhCNB.\u003c/p\u003e","manuscriptTitle":"Functional identification of the C-terminal domain of rhCNB","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 14:24:20","doi":"10.21203/rs.3.rs-7943596/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":"3501bccf-5808-4ce9-b120-c055344998db","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-04T10:11:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 14:24:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7943596","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7943596","identity":"rs-7943596","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}