Development of a CD39 nanobody and its enhancement to chimeric antigen receptor T cells efficacy against ovarian cancer in preclinical studies.

Rationale: CD39, a key ectonucleotidase that drives adenosine production, acts as a critical immunosuppressive checkpoint in cancer. Although it has shown promise as a therapeutic target, clinical trials are demonstrating the need for more potent targeting approaches. This need is driving innovation towards the development of novel antibodies and the exploration of strategic combinations with a range of immunotherapies. Methods: An anti-CD39 nanobody was screened and tested for its affinity and binding ability using biolayer interferometry, ELISA and flow cytometry. Blocking ability against soluble and membrane-bound CD39 was measured after CD39 blockade. Internalization was detected using immunofluorescence. The reversal of T-cell function by the anti-CD39 antibody was assessed by CFSE-based T-cell proliferation, CD25 expression and IFN-γ secretion. The in vivo function of tumor growth inhibition was further tested in a mouse model and we also tested the phenotype of immune cells after CD39 antibody administration from tumor tissue, draining lymph nodes and peripheral blood. We inserted the antibody sequence into the chimeric antigen receptor (CAR) construct to induce MSLN CAR-T cells to secret the CD39 antibody, and the efficacy was measured in xenograft models of ovarian cancer. Results: We screened human CD39 antibodies using a VHH library and developed a single-epitope anti-CD39 nanobody, named huCD39 mAb, with high affinity and potent binding and blocking ability. The huCD39 mAb was internalized in a time-dependent manner. The in vitro study revealed that the huCD39 mAb was highly effective in enhancing T-cell proliferation and functionality. In vivo, the huCD39 mAb showed significant anti-tumor efficacy in an immunocompetent mouse model. Flow cytometry analysis demonstrated downregulated CD39 expression in immune cells after antibody administration. We also observed increased CD39 expression in ovarian cancer tissue and in activated CAR T cells. Subsequently, we developed a type of MSLN CAR-T cells secreting huCD39 mAb which showed effective eradication or inhibition in ovarian tumor xenografts. Conclusions: A novel huCD39 mAb with strong blocking ability against human CD39 and potent inhibition of tumor growth has been developed. Furthermore, a modified huCD39 mAb-secreting CAR-T cell has been generated, exhibiting superior efficacy against ovarian cancer. This provides a promising strategy for optimizing immunotherapies in ovarian cancer and potentially other malignancies.

Combination therapy of Sanjie Zhentong capsules for endometriosis: Impact on hormone regulation and serum expression of FOLR1 and MSLN.

Examining the impact of Sanjie Zhentong capsules on hormone levels and serum expression of folate receptor 1 (FOLR1) and Mesothelin (MSLN) in endometriosis patients. A cohort of 150 endometriosis patients admitted between June 2019 and June 2023 was divided into 2 groups based on Sanjie Zhentong intake, comprising 75 cases each. Comparative analyses encompassed uterine artery hemodynamic parameters, ultrasound (B-mode) findings, Visual Analog Scale scores, traditional Chinese medicine symptom scores, Endometriosis Health Profile-30 scores, and serum levels of FOLR1, MSLN, and sex hormones before and after treatment. Post-treatment, both groups exhibited increased peak systolic velocity compared to pretreatment, with the Sanjie Zhentong group showing a significantly higher increase (P < .05). Moreover, resistance index decreased in both groups post-treatment, with the Sanjie Zhentong group displaying a greater reduction (P < .05). Adnexal mass diameters, endometrial thickness, and uterine volume decreased post-treatment in both groups, with the Sanjie Zhentong group experiencing a more pronounced decrease (P < .05). Serum FOLR1, MSLN, and sex hormone levels also decreased post-treatment in both groups, with the Sanjie Zhentong group exhibiting a more significant decrease (P < .05). Additionally, pain scores, traditional Chinese medicine symptoms, and Endometriosis Health Profile-30 scores decreased post-treatment in both groups, with the Sanjie Zhentong group demonstrating a greater reduction (P < .05). Sanjie Zhentong capsules demonstrate efficacy in regulating sex hormones, reducing serum FOLR1 and MSLN expression, alleviating pain, shrinking adnexal masses, decreasing uterine size, and improving uterine artery blood flow in endometriosis patients.

Microenvironmental alkalization promotes the therapeutic effects of MSLN-CAR-T cells.

Triple-negative breast cancer (TNBC) is characterized by high invasion, prone metastasis, frequent recurrence and poor prognosis. Unfortunately, the curative effects of current clinical therapies, including surgery, radiotherapy, chemotherapy and immunotherapy, are still limited in patients with TNBC. In this study, we showed that the heterogeneous expression at the protein level and subcellular location of mesothelin (MSLN), a potential target for chimeric antigen receptor-T (CAR-T) cell therapy in TNBC, which is caused by acidification of the tumor microenvironment, may be the main obstacle to therapeutic efficacy. Alkalization culture or sodium bicarbonate administration significantly promoted the membrane expression of MSLN and enhanced the killing efficiency of MSLN-CAR-T cells both in vitro and in vivo, and the same results were also obtained in other cancers with high MSLN expression, such as pancreatic and ovarian cancers. Moreover, mechanistic exploration revealed that the attenuation of autophagy-lysosome function caused by microenvironmental alkalization inhibited the degradation of MSLN. Hence, alkalization of the microenvironment improves the consistency and high expression of the target antigen MSLN and constitutes a routine method for treating diverse solid cancers via MSLN-CAR-T cells.

Mesothelin- and nucleolin-specific T cells from combined short peptides effectively kill triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC), known for its aggressiveness and limited treatment options, presents a significant challenge. Adoptive cell transfer, involving the ex vivo generation of antigen-specific T cells from peripheral blood mononuclear cells (PBMCs), emerges as a promising approach. The overexpression of mesothelin (MSLN) and nucleolin (NCL) in TNBC samples underscores their potential as targets for T cell therapy. This study explored the efficacy of multi-peptide pulsing of PBMCs to generate MSLN/NCL-specific T cells targeting MSLN+/NCL+ TNBC cells. METHODS: TNBC patient samples were confirmed for both MSLN and NCL expression via immunohistochemistry. Synthesized MSLN and NCL peptides were combined and administered to activate PBMCs from healthy donors. The cancer-killing ability of the resultant T cells was assessed using crystal violet staining, and their subtypes and cytotoxic cytokines were characterized through flow cytometry and cytokine bead array. RESULTS: Findings showed that 85.3% (127/149) of TNBC cases were positive for either MSLN or NCL, or both; with single positivity rates for MSLN and NCL of 14.1% and 28.9%, respectively. MSLN and NCL peptides, with high binding affinity for HLA-A*02, were combined and introduced to activated PBMCs from healthy donors. The co-pulsed PBMCs significantly induced TEM and TEMRA CD3+/CD8+ T cells and IFN-γ production, compared to single-peptide pulsed or unpulsed conditions. Notably, MSLN/NCL-specific T cells successfully induced cell death in MSLN+/NCL+ MDA-MB-231 cells, releasing key cytotoxic factors such as perforin, granzymes A and B, Fas ligand, IFN-γ, and granulysin. CONCLUSIONS: These findings serve as a proof-of-concept for using multiple immunogenic peptides as a novel therapeutic approach in TNBC patients.

Mesothelin expression correlates with elevated inhibitory immune activity in patients with colorectal cancer.

The expression of the protein Mesothelin (MSLN) is highly variable in several malignancies, including colorectal cancer (CRC), and high levels are associated with aggressive clinicopathological features and worse patient survival. Colorectal cancer is both a common and deadly cancer; being the third most common in incidence and second most common cause of cancer-related death. While systemic therapy remains the primary therapeutic option for most patients with stage IV (metastatic; m) CRC, their disease eventually becomes treatment refractory, and 85% succumb within 5 years. Microsatellite-stable (MSS) CRC tumors, which constitute more than 90% of patients with mCRC, are generally refractory to immunotherapeutic interventions. In our current work, we characterize MSLN levels in CRC, specifically correlating expression with clinical outcomes in relevant CRC subtypes, and explore how MSLN expression impacts the status of immune activation and suppression in the peritumoral microenvironment. Higher MSLN expression is prevalent in CMS1 and CMS4 CRC subtypes and correlates with higher gene mutation rates across the patient cohorts. Further, MSLN-high patients exhibit increased M1/M2 macrophage infiltration, PD-L1 staining, immune-inhibitory gene expression, enrichment in inflammatory, TGF-ß, IL6/JAK/STAT3, IL2/STAT5 signaling pathways, and mutation in KRAS and FBXW7. Together, these results suggest that MSLN protein is a potential target for antigen-specific therapy and supports investigation into its tumorigenic effects to identify possible therapeutic interventions for patients with high MSLN expressing MSS CRC.

Phosphodiesterase-5 inhibition collaborates with vaccine-based immunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistant to immunotherapy. Although immune recognition can be enhanced with immunomodulatory agents including checkpoint inhibitors and vaccines, few patients experience clinical efficacy because the tumor immune microenvironment (TiME) is dominated by immunosuppressive myeloid cells that impose T cell inhibition. Inhibition of phosphodiesterase-5 (PDE5) was reported to downregulate metabolic regulators arginase and inducible NOS in immunosuppressive myeloid cells and enhance immunity against immune-sensitive tumors, including head and neck cancers. We show for the first time to our knowledge that combining a PDE5 inhibitor, tadalafil, with a mesothelin-specific vaccine, anti-programmed cell death protein 1, and anti-cytotoxic T lymphocyte-associated protein 4 yields antitumor efficacy even against immune-resistant PDAC. To determine immunologic advantages conferred by tadalafil, we profiled the TiME using mass cytometry and single-cell RNA-sequencing analysis with Domino to infer intercellular signaling. Our analyses demonstrated that tadalafil reprograms myeloid cells to be less immunosuppressive. Moreover, tadalafil synergized with the vaccine, enhancing T cell activation including mesothelin-specific T cells. Tadalafil treatment was also associated with myeloid/T cell signaling axes important for antitumor responses (e.g., Cxcr3, Il12). Our study shows that PDE5 inhibition combined with vaccine-based immunotherapy promotes pro-inflammatory states of myeloid cells, activation of T cells, and enhanced myeloid/T cell crosstalk to yield antitumor efficacy against immune-resistant PDAC.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclin B1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosis I. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphase I (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclin B1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphase II (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

Randomized trial of anetumab ravtansine and pembrolizumab compared to pembrolizumab for mesothelioma.

PURPOSE: The mesothelin-targeting antibody-drug conjugate anetumab ravtansine was evaluated in combination with the programmed cell death-1 (PD-1) inhibitor pembrolizumab based on the common expression of mesothelin and reports of activity in mesothelioma. PATIENTS AND METHODS: A phase 1 safety run-in of the combination of anetumab ravtansine (6.5 mg/kg iv q3weeks) and pembrolizumab (200 mg, IV q3weeks) was conducted, followed by a phase 2 randomization to the combination or pembrolizumab alone at medical centers across the United States and Canada in the National Cancer Institute's Experimental Therapeutics Clinical Trials Network. Patients with pleural mesothelioma that expressed mesothelin and had previously received platinum-based therapy were eligible. RESULTS: In phase 1 (n = 12) only one dose limiting toxicity was observed and the rules for dose reduction were not met. In phase 2, there was no difference in the confirmed response rates between the combination group (n = 18, 2 partial responses [PR], 11 %) and the pembrolizumab group (n = 17, 1 PR, 6 %; z = -0.5523, p = 0.29116). The median PFS was 12.2 months (95 % CI 5.1-not evaluable [NE]) for the combination, and 3.9 months for pembrolizumab (95 % CI 2.1-NE)(HR=0.55, p = 0.20). Patients with high baseline levels of soluble mesothelin who received anetumab ravtansine had a median PFS of 5 months. CONCLUSIONS: The numeric difference in PFS between treatment groups was not statistically significant, likely related to a smaller than planned sample size. High levels of soluble mesothelin should potentially be considered to select against the use of mesothelin-targeting therapies in development that are neutralized by soluble mesothelin.

Mesothelin promotes the migration of endometrioid carcinoma and is associated with the MELF pattern.

Mesothelin (MSLN) is expressed in the mesothelium in normal tissues but is overexpressed in various malignant tumors. In this study, we searched for genes that were more frequently expressed in cases of endometrioid carcinoma (EC) with the MELF (microcystic, elongated, and fragmented) pattern using laser microdissection and RNA sequencing, and found that MSLN was predominantly expressed in cases with the MELF pattern. The role of MSLN in EC was analyzed by generating MSLN-knockout and -knockdown EC cell lines. MSLN promoted migration and epithelial-mesenchymal transition (EMT). Moreover, we found that cadherin-6 (CDH6) expression was regulated by MSLN. MSLN is known to bind to cancer antigen 125 (CA125), and we found that CA125 can regulate CDH6 expression via MSLN. Immunohistochemical investigations showed that MSLN, CA125, and CDH6 expression levels were considerably elevated in EC with the MELF pattern. The expression of CA125 was similar to that of MSLN not only in terms of immunohistochemical staining intensity but also the blood level of CA125. Our results showed that MSLN contributes to the migration and EMT of EC cells through upstream CA125 and downstream CDH6. Therefore, MSLN has potential as a therapeutic target for EC with the MELF pattern.

CAR memory-like NK cells targeting the membrane proximal domain of mesothelin demonstrate promising activity in ovarian cancer.

Epithelial ovarian cancer (EOC) remains one of the most lethal gynecological cancers. Cytokine-induced memory-like (CIML) natural killer (NK) cells have shown promising results in preclinical and early-phase clinical trials. In the current study, CIML NK cells demonstrated superior antitumor responses against a panel of EOC cell lines, increased expression of activation receptors, and up-regulation of genes involved in cell cycle/proliferation and down-regulation of inhibitory/suppressive genes. CIML NK cells transduced with a chimeric antigen receptor (CAR) targeting the membrane-proximal domain of mesothelin (MSLN) further improved the antitumor responses against MSLN-expressing EOC cells and patient-derived xenograft tumor cells. CAR arming of the CIML NK cells subtanstially reduced their dysfunction in patient-derived ascites fluid with transcriptomic changes related to altered metabolism and tonic signaling as potential mechanisms. Lastly, the adoptive transfer of MSLN-CAR CIML NK cells demonstrated remarkable inhibition of tumor growth and prevented metastatic spread in xenograft mice, supporting their potential as an effective therapeutic strategy in EOC.

Impact of fludarabine and treosulfan on ovarian tumor cells and mesothelin chimeric antigen receptor T cells.

In addition to their immunosuppressive effect, cytostatics conditioning prior to adoptive therapy such as chimeric antigen receptor (CAR) T cells may play a role in debulking and remodeling the tumor microenvironment. We investigated in vitro the killing efficacy and impact of treosulfan and fludarabine on ovarian cancer cells expressing mesothelin (MSLN) and effect on MSLN-targeting CAR T cells. Treosulfan and fludarabine had a synergetic effect on killing of SKOV3 and OVCAR4 cells. Sensitivity to the combination of treosulfan and fludarabine was increased when SKOV3 cells expressed MSLN and when OVCAR4 cells were tested in hypoxia, while MSLN cells surface expression by SKOV3 and OVCAR4 cells was not altered after treosulfan or fludarabine exposure. Exposure to treosulfan or fludarabine (10 µM) neither impacted MSLN-CAR T cells degranulation, cytokines production upon challenge with MSLN + OVCAR3 cells, nor induced mitochondrial defects. Combination of treosulfan and fludarabine decreased MSLN-CAR T cells anti-tumor killing in normoxia but not hypoxia. In conclusion, treosulfan and fludarabine killed MSLN + ovarian cancer cells without altering MSLN-CAR T cells functions (at low cytostatics concentration) even in hypoxic conditions, and our data support the use of treosulfan and fludarabine as conditioning drugs prior to MSLN-CAR T cell therapy.

Proton radiation boosts the efficacy of mesothelin-targeting chimeric antigen receptor T cell therapy in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents a challenge in oncology, with limited treatment options for advanced-stage patients. Chimeric antigen receptor T cell (CAR T) therapy targeting mesothelin (MSLN) shows promise, but challenges such as the hostile immunosuppressive tumor microenvironment (TME) hinder its efficacy. This study explores the synergistic potential of combining proton radiation therapy (RT) with MSLN-targeting CAR T therapy in a syngeneic PDAC model. Proton RT significantly increased MSLN expression in tumor cells and caused a significant increase in CAR T cell infiltration into tumors. The combination therapy reshaped the immunosuppressive TME, promoting antitumorigenic M1 polarized macrophages and reducing myeloid-derived suppressor cells (MDSC). In a flank PDAC model, the combination therapy demonstrated superior attenuation of tumor growth and improved survival compared to individual treatments alone. In an orthotopic PDAC model treated with image-guided proton RT, tumor growth was significantly reduced in the combination group compared to the RT treatment alone. Further, the combination therapy induced an abscopal effect in a dual-flank tumor model, with increased serum interferon-γ levels and enhanced proliferation of extratumoral CAR T cells. In conclusion, combining proton RT with MSLN-targeting CAR T therapy proves effective in modulating the TME, enhancing CAR T cell trafficking, and exerting systemic antitumor effects. Thus, this combinatorial approach could present a promising strategy for improving outcomes in unresectable PDAC.

rhIL-7-hyFc, a long-acting interleukin-7, improves efficacy of CAR-T cell therapy in solid tumors.

BACKGROUND: Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy approach using an engineered long-acting interleukin (IL)-7 (rhIL-7-hyFc or NT-I7) and CAR-T cells targeting three antigens, glypican-2 (GPC2), glypican-3 (GPC3), and mesothelin (MSLN), against multiple solid tumor types including liver cancer, neuroblastoma, ovarian cancer, and pancreatic cancer in mice. METHODS: CAR-T cells targeting GPC2, GPC3, and MSLN were used in combination with NT-I7 to assess the anticancer activity. Xenograft tumor models, including the liver cancer orthotopic model, were established using NOD scid gamma mice engrafted with cell lines derived from hepatocellular carcinoma, neuroblastoma, ovarian cancer, and pancreatic cancer. The mice were monitored by bioluminescence in vivo tumor imaging and tumor volume measurement using a caliper. Immunophenotyping of CAR-T cells on NT-I7 stimulation was evaluated for memory markers, exhaust markers, and T-cell signaling molecules by flow cytometry and western blotting. RESULTS: Compared with the IL-2 combination, preclinical evaluation of NT-I7 exhibited regression of solid tumors via enhanced occupancy of CD4+ CAR-T, improved T-cell expansion, reduced exhaustion markers (programmed cell death protein 1 or PD-1 and lymphocyte-activation gene 3 or LAG-3) expression, and increased generation of stem cell-like memory CAR-T cells. The STAT5 pathway was demonstrated to be downstream of NT-I7 signaling, mediated by increased expression of the IL-7 receptor expression in CAR-T cells. Furthermore, CAR-T cells improved efficacy against tumors with low antigen density when combined with NT-I7 in mice, presenting an avenue for patients with heterogeneous antigenic profiles. CONCLUSION: This study provides a rationale for NT-I7 plus CAR-T cell combination therapy for solid tumors in humans.

T cell-redirecting antibody for treatment of solid tumors via targeting mesothelin.

T cell engaging bispecific antibodies (TCBs) have recently become significant in cancer treatment. In this study we developed MSLN490, a novel TCB designed to target mesothelin (MSLN), a glycosylphosphatidylinositol (GPI)-linked glycoprotein highly expressed in various cancers, and evaluated its efficacy against solid tumors. CDR walking and phage display techniques were used to improve affinity of the parental antibody M912, resulting in a pool of antibodies with different affinities to MSLN. From this pool, various bispecific antibodies (BsAbs) were assembled. Notably, MSLN490 with its IgG-[L]-scFv structure displayed remarkable anti-tumor activity against MSLN-expressing tumors (EC50: 0.16 pM in HT-29-hMSLN cells). Furthermore, MSLN490 remained effective even in the presence of non-membrane-anchored MSLN (soluble MSLN). Moreover, the anti-tumor activity of MSLN490 was enhanced when combined with either Atezolizumab or TAA × CD28 BsAbs. Notably, a synergistic effect was observed between MSLN490 and paclitaxel, as paclitaxel disrupted the immunosuppressive microenvironment within solid tumors, enhancing immune cells infiltration and improved anti-tumor efficacy. Overall, MSLN490 exhibits robust anti-tumor activity, resilience to soluble MSLN interference, and enhanced anti-tumor effects when combined with other therapies, offering a promising future for the treatment of a variety of solid tumors. This study provides a strong foundation for further exploration of MSLN490's clinical potential.

Fangchinoline inhibits mouse oocyte meiosis by disturbing MPF activity.

Fangchinoline (FA) is an alkaloid derived from the traditional Chinese medicine Fangji. Numerous studies have shown that FA has a toxic effect on various cancer cells, but little is known about its toxic effects on germ cells, especially oocytes. In this study, we investigated the effects of FA on mouse oocyte maturation and its potential mechanisms. Our results showed that FA did not affect meiosis resumption but inhibited the first polar body extrusion. This inhibition is not due to abnormalities at the organelle level, such as chromosomes and mitochondrial, which was proved by detection of DNA damage and reactive oxygen species. Further studies revealed that FA arrested the oocyte at the metaphase I stage, and this arrest was not caused by abnormal kinetochore-microtubule attachment or spindle assembly checkpoint activation. Instead, FA inhibits the activity of anaphase-promoting complexes (APC/C), as evidenced by the inhibition of CCNB1 degeneration. The decreased activity of APC/C may be due to a reduction in CDC25B activity as indicated by the high phosphorylation level of CDC25B (Ser323). This may further enhance Maturation-Promoting Factor (MPF) activity, which plays a critical role in meiosis. In conclusion, our study suggests that the metaphase I arrest caused by FA may be due to abnormalities in MPF and APC/C activity.

Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.

A mesothelin microsensor based on an embedded thionine electronic medium within an imprinted polymer on an acupuncture needle electrode.

An electrochemical microsensor for mesothelin (MSLN) based on an acupuncture needle (AN) was constructed in this work. To prepare the microsensor, MSLN was self-assembled on 4-mercaptophenylboronic acid (4-MPBA) by an interaction force between the external cis-diol and phenylboronic acid. This was followed by the gradual electropolymerization of thionine (TH) and eriochrome black T (EBT) around the anchored protein. The thickness of the surface imprinted layers influenced the sensing performance and needed to be smaller than the height of the anchored protein. The polymerized EBT was not electrically active, but the polymerized TH provided a significant electrochemical signal. Therefore, electron transfer smoothly proceeded through the eluted nanocavities. The imprinted nanocavities were highly selective toward MSLN, and the rebinding of insulating proteins reduced the electrochemical signal of the embedded pTH. The functionalized interface was characterized by SEM and electrochemical methods, and the preparation conditions were studied. After optimization, the sensor showed a linear response in the range of 0.1 to 1000 ng mL-1 with a detection limit of 10 pg mL-1, indicating good performance compared with other reported methods. This microsensor also showed high sensitivity and stability, which can be attributed to the fine complementation of the imprinted organic nanocavities. The sensitivity of this sensor was related to the nanocavities used for electron transport around the AuNPs. In the future, microsensors that can directly provide electrochemical signals are expected to play important roles especially on AN matrices.

CD8+ T cell targeting of tumor antigens presented by HLA-E.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.