Targeting MutT Homolog 1 (MTH1) for Breast Cancer Suppression by Using a Novel MTH1 Inhibitor MA-24 with Tumor-Selective Toxicity.

BACKGROUND: Breast cancer is a commonly diagnosed cancer worldwide. Human MutT homolog 1 (MTH1) is found to be elevated in breast tumors and cancer cells need MTH1 for survival. Pharmacological inhibition of MTH1 may be potentially beneficial in the treatment of breast cancer. METHODS: MA-24 was screened by malachite green colorimetric assay for MTH1 inhibitors and the kinetic characteristics of MA-24 were assessed. The features of MA-24's binding with MTH1 were ascertained through molecular docking, and the cytotoxic activity of MA-24 was validated in vitro and in vivo. Target engagement assays, comet assay, and Western blot confirmed the intracellular target and mechanism of MA-24. RESULTS: MA-24 shows potent antitumor bioactivity both in vitro and in vivo. MA-24 competitively inhibited the MTH1 and further induced DNA strand breaks, leading to increased apoptosis of cancer cells depending on the upregulation of the cleaved-caspase 3-cleaved-PARP axis. In particular, MA-24 exhibited a powerful efficacy and safety in vivo (tumor growth inhibition rate: 61.8%). CONCLUSIONS: MA-24 possesses a broad spectrum of breast cancer cytotoxicity and offered valuable insights for overcoming the challenges of chemotherapy-related toxicity, which holds great potential for the further development MA-24 as an anti-cancer drug.

Novel specific anti-ESM1 antibodies overcome tumor bevacizumab resistance by suppressing angiogenesis and metastasis.

Suppressing tumors through anti-angiogenesis has been established as an effective clinical treatment strategy. Bevacizumab, a monoclonal antibody, is commonly used in various indications. However, two major challenges limit the long-term efficacy of bevacizumab: drug resistance and side effects. Bevacizumab resistance has been extensively studied at the molecular level, but no drug candidates have been developed for clinical use to overcome this resistance. In a previous study conducted by our team, a major finding was that high expression of ESM1 in bevacizumab-resistant tumors is associated with an unfavorable response to treatment. In particular, an increase in ESM1 expression contributes to heightened lung metastasis and microvascular density, which ultimately decreases the tumor's sensitivity to bevacizumab. In contrast, the silencing of ESM1 results in reduced angiogenesis and suppressed tumor growth in tumors resistant to bevacizumab. We put forward the hypothesis that targeting ESM1 could serve as a therapeutic strategy in overcoming bevacizumab resistance. In this study, a variety of anti-ESM1 antibodies with high affinity to human ESM1 were successfully prepared and characterized. Our in vivo study confirmed the establishment of a bevacizumab-resistant human colorectal cancer model and further demonstrated that the addition of anti-ESM1 monoclonal antibodies to bevacizumab treatment significantly improved tumor response while downregulating DLL4 and MMP9. In conclusion, our study suggests that anti-hESM1 monoclonal antibodies have the potential to alleviate or overcome bevacizumab resistance, thereby providing new strategies and drug candidates for clinical research in the treatment of bevacizumab-resistant colorectal cancer.

Endothelial-Specific Molecule 1 Inhibition Lessens Productive Angiogenesis and Tumor Metastasis to Overcome Bevacizumab Resistance.

The development of drug resistance in malignant tumors leads to disease progression, creating a bottleneck in treatment. Bevacizumab is widely used clinically, and acts by inhibiting angiogenesis to "starve" tumors. Continuous treatment can readily induce rebound proliferation of tumor blood vessels, leading to drug resistance. Previously, we found that the fragment crystallizable (Fc) region of bevacizumab cooperates with the Toll-like receptor-4 (TLR4) ligand to induce M2b polarization in macrophages and secrete tumor necrosis factor-α (TNFα), which promotes immunosuppression, tumor metastasis, and angiogenesis. However, the downstream mechanism underlying TNFα-mediated bevacizumab resistance requires further investigation. Our RNA-Seq analysis results revealed that the expression of endothelial cell specific molecule-1 (ESM1) increased significantly in drug-resistant tumors and promoted metastasis and angiogenesis in vitro and in vivo. Furthermore, TNFα induced the upregulation of ESM1, which promotes metastasis and angiogenesis and regulates matrix metalloprotease-9 (MMP9), vascular endothelial growth factor (VEGF), and delta-like ligand-4 molecules (DLL4). Accordingly, the curative effect of bevacizumab improved by neutralizing ESM1 with high-affinity anti-ESM1 monoclonal antibody 1-2B7 in bevacizumab-resistant mice. This study provides important insights regarding the molecular mechanism by which TNFα-induced ESM1 expression promotes angiogenesis, which is significant for elucidating the mechanism of bevacizumab drug resistance and possibly identifying appropriate biosimilar molecules.

A novel lncRNA lncSAMD11-1: 1 interacts with PIP4K2A to promote endometrial decidualization by stabilizing FoxO1 nuclear localization.

Decidualization is essential for a successful pregnancy and determines embryo implantation and pregnancy maintenance. Abnormal decidualization is one of the main causes of recurrent implantation failure (RIF). Studies have shown that large amounts of long noncoding RNAs (lncRNAs) are abnormally expressed in endometrial samples from patients with RIF. However, the functional contributions of lncRNAs to decidualization in RIF have not been explored. In this study, we found that lncSAMD11-1:1 was significantly declined in the endometria of patients with RIF. The knockdown of lncSAMD11-1:1 in human endometrial stromal cells (hESCs) restrained decidualization and embryo implantation in vitro, while the overexpression of lncSAMD11-1:1 facilitated hESC decidualization and embryo implantation in vitro and ameliorated decidualization in RIF patients. Mechanistically, lncSAMD11-1:1 and phosphatidylinositol-5-phosphate 4-kinase type 2 alpha (PIP4K2A) translocated out of nucleus and bound to each other during decidualization, thereby inhibiting the phosphorylation of AKT and promoting FoxO1 nuclear localization. These data suggest that lncSAMD11-1:1 might be a critical novel lncRNA functionally required for human decidualization, and the dysregulation of lncSAMD11-1:1 in the endometrium may be a new predisposing factor of RIF.

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research.

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

Calpain7 negatively regulates human endometrial stromal cell decidualization in EMs by promoting FoxO1 nuclear exclusion via hydrolyzing AKT1.

Endometrial receptivity damage caused by impaired decidualization may be one of the mechanisms of infertility in endometriosis (EMs). Our previous study demonstrated that Calpain-7 (CAPN7) is abnormally overexpressed in EMs. Whether CAPN7 affects the regulation of decidualization and by what mechanism CAPN7 regulates decidualization remains to be determined. In this study, we found CAPN7 expression decreased during human endometrial stromal cell (HESC) decidualization in vitro. CAPN7 negatively regulated decidualization in vitro and in vivo. We also identified one conserved potential PEST sequence in the AKT1 protein and found that CAPN7 was able to hydrolyse AKT1 and enhance AKT1's phosphorylation. Correspondingly, CAPN7 notably promoted the phosphorylation of Forkhead Box O1 (FoxO1), the downstream of AKT1 protein, at Ser319, leading to increased FoxO1 exclusion from nuclei and attenuated FoxO1 transcriptional activity in decidualized HESC. In addition, we detected endometrium CAPN7, p-AKT1, and p-FoxO1 expressions were increased in EMs. These data demonstrate that CAPN7 negatively regulates HESC decidualization in EMs probably by promoting FoxO1's phosphorylation and FoxO1 nuclear exclusion via hydrolyzing AKT1. The dysregulation of CAPN7 may be a novel cause of EMs.

Transcriptome analysis of eutopic endometrium in adenomyosis after GnRH agonist treatment.

BACKGROUND: Adenomyosis is a chronic gynecological disease characterized by invasion of the uterine endometrium into the muscle layer. In assisted reproductive technology (ART), gonadotropin-releasing hormone agonist (GnRHa) is often used to improve pregnancy rates in patients with adenomyosis, but the underlying mechanisms are poorly understood. METHODS: Eutopic endometrial specimens were collected from patients with adenomyosis before and after GnRHa treatment in the midsecretory phase. RNA sequencing (RNA-Seq) of these specimens was performed for transcriptome analysis. The differentially expressed genes (DEGs) of interest were confirmed by real-time PCR and immunohistochemistry. RESULTS: A total of 132 DEGs were identified in the endometrium of patients with adenomyosis after GnRHa treatment compared with the control group. Bioinformatics analysis predicted that immune system-associated signal transduction changed significantly after GnRHa treatment. Chemokine (C-C motif) ligand 21 (CCL21) was found to be highly expressed in the eutopic endometrium after GnRHa treatment, which may be involved in the improvement of endometrial receptivity in adenomyosis. CONCLUSION: This study suggests that molecular regulation related to immune system-associated signal transduction is an important mechanism of GnRHa treatment in adenomyosis. Immunoreactive CCL21 is thought to regulate inflammatory events and participate in endometrial receptivity in adenomyosis.

Attenuated monoamine oxidase a impairs endometrial receptivity in women with adenomyosis via downregulation of FOXO1†.

The establishment of endometrial receptivity is a prerequisite for successful pregnancy. Women with adenomyosis possess a lower chance of clinical pregnancy after assisted reproductive technology, which is partially due to impaired endometrial receptivity. The establishment of endometrial receptivity requires the participation of multiple processes, and proper endometrial epithelial cell (EEC) proliferation is indispensable. Monoamine oxidase A (MAOA) is a key molecule that regulates neurotransmitter metabolism in the nervous system. In the present study, we demonstrated a novel role for MAOA in the establishment of endometrial receptivity in women with adenomyosis and in an adenomyotic mouse model. Attenuated MAOA impairs endometrial receptivity by promoting inappropriate proliferation of EECs via the downregulation of FOXO1 during the window of implantation. These results revealed that MAOA plays a vital role in endometrial receptivity in female reproduction.

The Value of MR-DWI and T1 Mapping in Indicating Radiation-Induced Soft Tissue Injury.

OBJECTIVE: To explore the value of MR-DWI and T1 mapping in predicting radiation-induced soft tissue fibrosis and its correlation with radiation inflammation. METHODS: ① a total of 30 C57BL/6 mice were randomly divided into a control group (Nor group), irradiation group (IR group) and irradiation plus glycyrrhetinic acid group (GA group). The IR group and GA group were treated with 6MV X-rays to irradiate the right hind limbs of mice for 30 Gy in a single shot. MRI examinations were performed before and on the 7th day after irradiation to measure the apparent diffusion coefficient (ADC) value and the longitudinal relaxation time (T1) value of the hind limb muscles of the mice. On the 90th day after irradiation, the hind limb contracture was measured, and the right hind limb muscle was taken for HE staining, masson staining, immunohistochemical staining and Western blot analysis to detect the expression of a-SMA and Fibronectin. ② The other 30 mice were grouped randomly as above. On the 7th day after irradiation, the right hind limbs of the mice were examined by MRI to measure the ADC value and T1 value of the thigh muscles, and then the right hind thigh muscles were immediately sacrificed to detect IL-1ß, IL-6, TNF-a and TGF-ß1 expression with ELISA. RESULTS: On the 7th day after irradiation, the ADC values ​​of right hind thigh muscles of mice in Nor group, IR group and GA group were (1.35 ± 0.11)*10-3mm2/s, (1.48 ± 0.07) *10-3mm2/s and (1.36 ± 0.13)*10-3mm2/s, respectively, by which the differences between the IR group and Nor group (P=0.008) and that between IR group and GA group (P=0.013) were statistically significant; T1 values ​​were (1369.7 ± 62.7)ms, (1483.7 ± 127.7)ms and (1304.1 ± 82.3)ms, respectively, with which the differences in the T1 value between the IR group and Nor group (P=0.012) and between IR group and GA group (P<0.001) were also statistically significant. On the 90th day after irradiation, the contracture lengths of the right hind limbs of the three groups of mice were (0.00 ± 0.07)cm, (2.08 ± 0.32)cm, and (1.49 ± 0.70) cm, respectively. There were statistically significant differences in the IR group compared with the Nor group (P<0.001) and the GA group (P=0.030). The ADC value (r=0.379, P=0.039) and T1 value (r=0.377, P=0.040) of the mice's hindlimbs on Day 7 after irradiation were correlated with the degree of contracture on Day 90 after irradiation; the ADC value (r=0.496, P=0.036) and T1 value (r=0.52, P=0.027) were positively correlated with the Masson staining results and with the expression of α-SMA and Fibronectin. While the ADC value was positively correlated with IL-6 (r=0.553, P=0.002), there was no obvious correlation with IL-1ß, TNF-a and TGF-ß1; the T1 value was positively correlated with IL-1ß (r=0.419, P=0.021), IL-6 (r=0.535, P=0.002) and TNF-a (r=0.540, P=0.002) but not significantly related to TGF-ß1 (r=0.155, P=0.413). CONCLUSION: The MR-DWI and T1 mapping values on the 7th day after irradiation can reflect the early condition of tissue inflammation after the soft tissue is irradiated, and the values have a certain correlation with the degree of radiofibrosis of the soft tissue in the later period and may be used as an index to predict radiofibrosis.

Tumor necrosis factor α inhibition overcomes immunosuppressive M2b macrophage-induced bevacizumab resistance in triple-negative breast cancer.

Bevacizumab in neoadjuvant therapy provides a new hope of improved survival for patients with triple-negative breast cancer (TNBC) by targeting vascular endothelial growth factor in combination with chemotherapy, but curative effect is limited by bevacizumab's continuous use while mechanisms remain incompletely understood. More and more researches reported that tumor-associated macrophages mediate resistance to chemotherapy and radiotherapy in various tumors. Here we developed a TNBC model resistant to bevacizumab under bevacizumab continuous administration. It was found that proportion of a specific subset of tumor-associated macrophages characterized as M2b (CD11b+ CD86high IL10high) increased and responsible for acquired resistance to bevacizumab. Then, we showed that RAW264.7 macrophages could be polarized to M2b subtype on simultaneous exposure to bevacizumab and TLR4 ligands as occurs in the context of continuous bevacizumab treatment. Concordantly, in TLR4-deleted C57BL/10ScNJNju (TLR4lps-del) mut/mut mice with bevacizumab treatment model, it was verified that the M2b macrophage could be induced by Fc gamma receptor-TLR4 cross-talk. In MDA-MB-231-resistant tumor-bearing mice, the content of TNFα in serum kept going up consistent with CCL1, a chemokine of M2b macrophage. In vitro neutralizing tumor necrosis factor α (TNFα) could inhibit the tumor progression caused by M2b culture medium and tumor IDO1 expression. Therefore, we thought that TNFα is a key tumor-promoting effector molecule secreted by M2b macrophage. Accordingly, the curative effect of bevacizumab was proved to be significantly improved by neutralizing TNFα with anti-TNFα nanobody. This study is expected to provide theoretical and clinical evidence elucidating the drug resistance in patients receiving bevacizumab.

Preparation of RGD4C fused anti-TNFα nanobody and inhibitory activity on triple-negative breast cancer in vivo.

AIMS: Triple-negative breast cancer (TNBC) is not sensitive to current endocrine treatments, so new treatment strategies need to be explored. Based on previous antitumour studies on anti-TNFα nanobody, we designed a novel fusion nanobody to enhance antitumour activity of the anti-TNFα nanobody in TNBC. MAIN METHODS: The RGD4C contains RGD sequence, which is the smallest recognition unit binding to the αvß3 receptor on tumour cell membranes and involved in tumour cell adhesion, proliferation, and metastasis. RGD4C was fused to anti-TNFα nanobody to investigate the antitumour activity in vitro and in vivo. KEY FINDINGS: The antitumour effects of fusion nanobody V-L-R-H could effectively bind to αvß3 and inhibit cell migration and proliferation of MDA-MB-231, which had satisfying purification efficiency and approving antigen or receptor binding activity. V-L-R-H could inhibit the TNFα-mediated PI3K/AKT/NF-κB signal pathway and integrin αvß3 correlative FAK focal adhesion signal pathway. Mouse xenograft tumour experiments showed that the V-L-R-H could inhibit tumour proliferation and metastasis; reduce the TNFα, HIFα, Ki67, and CD31 concentrations in tumour; and inhibit the process of epithelial-mesenchymal transition. SIGNIFICANCE: The fusion nanobody enhanced antitumour activity of the anti-TNFα nanobody on TNBC. It provided a reference for the design of dual functional fusion proteins and development of tumour treatment strategies of antagonistic TNFα and αvß3, and a new therapeutic strategy and research direction for the treatment of TNBC.

Performance of diffusion and perfusion MRI in evaluating primary central nervous system lymphomas of different locations.

BACKGROUND: Diffusion and perfusion MRI can invasively define physical properties and angiogenic features of tumors, and guide the individual treatment. The purpose of this study was to investigate whether the diffusion and perfusion MRI parameters of primary central nervous system lymphomas (PCNSLs) are related to the tumor locations. METHODS: We retrospectively reviewed the diffusion, perfusion, and conventional MRI of 68 patients with PCNSLs at different locations (group 1: cortical gray matter, group 2: white matter, group 3: deep gray matter). Relative maximum cerebral blood volume (rCBVmax) from perfusion MRI, minimum apparent diffusion coefficients (ADCmin) from DWI of each group were calculated and compared by one-way ANOVA test. In addition, we compared the mean apparent diffusion coefficients (ADCmean) in three different regions of control group. RESULTS: The rCBVmax of PCNSLs yielded the lowest value in the white matter group, and the highest value in the cortical gray matter group (P < 0.001). However, the ADCmin of each subgroup was not statistically different. The ADCmean of each subgroup in control group was not statistically different. CONCLUSION: Our study confirms that rCBVmax of PCNSLs are related to the tumor location, and provide simple but effective information for guiding the clinical practice of PCNSLs.

MicroRNA-181a promotes follicular granulosa cell apoptosis via sphingosine-1-phosphate receptor 1 expression downregulation†.

Oxidative stress induces granulosa cell (GC) apoptosis and subsequent follicular atresia. Since our previous studies indicate that microRNA-181a (miR-181a) expression is increased in GCs undergoing apoptosis, the present study was designed to define the relationship between exposure to oxidative stressors in GCs and changes in miR-181a expression and function. To achieve this, we employed an H2O2-induced in vitro model and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. We demonstrated that in vitro miR-181a overexpression promoted GC apoptosis in a dose-dependent manner; sphingosine-1-phosphate (S1P) significantly reversed both H2O2-induced and miR-181a-induced apoptosis in GCs. Moreover, we identified sphingosine-1-phosphate receptor 1 (S1PR1), a critical receptor of S1P, as a novel target of miR-181a in GCs. MicroRNA-181a induced GC apoptosis by repressing S1PR1 expression in vitro. Importantly, increased miR-181a expression and decreased S1PR1 expression were detected in the in vivo ovarian oxidative stress model by Western blot analysis and immunohistochemistry. Furthermore, we found similar expression patterns of miR-181a and S1PR1 in GCs from patients with premature ovarian insufficiency. In conclusion, our results suggest that miR-181a directly suppresses expression of S1PR1, which has critical roles in mediating oxidative stress-induced GC apoptosis both in vitro and in vivo.

A Novel Specific Anti-CD73 Antibody Inhibits Triple-Negative Breast Cancer Cell Motility by Regulating Autophagy.

Increasing researches have focused on cancer metastasis and development. The ectonucleotidase CD73 is one of the most common cell surface enzymes that are involved in immunosuppression. In this study, the recombinant plasmid pET28a-CD73 was constructed and the CD73 protein was overexpressed in E. coli as an inclusion body that was then subjected to refolding. The anti-CD73 monoclonal antibody (3F7) was obtained by hybridoma technology. The antibody subtype was identified as IgG2a with an affinity constant of 5.75 nM. This antibody could be applied to immunofluorescence and flow cytometry. The results showed that the CD73 protein was not only located in the cytoplasm but also distributed on the surface of triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468. Moreover, the level of CD73 protein was associated with the survival rate. Although the anti-CD73 antibody was not able to inhibit tumor cell growth, it could enhance the cytotoxic effect of Doxorubicin to triple-negative breast cancer cells. In vitro function assay results indicated that anti-CD73 mAb could inhibit cell migration and invasion in both human triple-negative breast cancer and mouse 4T1 cell lines. In this process, both the LC3I/LC3II ratio and p62 protein levels increased, which indicated that the blockage of CD73 could inhibit cell autophagy, and cell migration and invasion were restored by rapamycin. In vivo, anti-CD73 mAb could significantly inhibit lung metastasis of 4T1 cells in a mouse xenograft model. Taken together, this novel anti-CD73 antibody could be developed as an adjuvant drug for triple-negative breast cancer therapy and can be useful in tumor diagnosis.

Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target.

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvß5 (ITGαvß5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB-OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumour­targeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an Ni­NTA column and a G­50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by high­performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec­1, respectively. The anti­hMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF­7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.

Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.