Goliath induces inflammation in obese mice by linking fatty acid ß-oxidation to glycolysis.

Obesity is associated with metabolic disorders and chronic inflammation. However, the obesity-associated metabolic contribution to inflammatory induction remains elusive. Here, we show that, compared with lean mice, CD4+ T cells from obese mice exhibit elevated basal levels of fatty acid ß-oxidation (FAO), which promote T cell glycolysis and thus hyperactivation, leading to enhanced induction of inflammation. Mechanistically, the FAO rate-limiting enzyme carnitine palmitoyltransferase 1a (Cpt1a) stabilizes the mitochondrial E3 ubiquitin ligase Goliath, which mediates deubiquitination of calcineurin and thus enhances activation of NF-AT signaling, thereby promoting glycolysis and hyperactivation of CD4+ T cells in obesity. We also report the specific GOLIATH inhibitor DC-Gonib32, which blocks this FAO-glycolysis metabolic axis in CD4+ T cells of obese mice and reduces the induction of inflammation. Overall, these findings establish a role of a Goliath-bridged FAO-glycolysis axis in mediating CD4+ T cell hyperactivation and thus inflammation in obese mice.

Rescue of cardiac dysfunction during chemotherapy in acute myeloid leukaemia by blocking IL-1α.

BACKGROUND AND AIMS: Patients with acute myeloid leukaemia (AML) suffer from severe myocardial injury during daunorubicin (DNR)-based chemotherapy and are at high risk of cardiac mortality. The crosstalk between tumour cells and cardiomyocytes might play an important role in chemotherapy-related cardiotoxicity, but this has yet to be demonstrated. This study aimed to identify its underlying mechanism and explore potential therapeutic targets. METHODS: Cardiac tissues were harvested from an AML patient after DNR-based chemotherapy and were subjected to single-nucleus RNA sequencing. Cardiac metabolism and function were evaluated in AML mice after DNR treatment by using positron emission tomography, magnetic resonance imaging, and stable-isotope tracing metabolomics. Plasma cytokines were screened in AML mice after DNR treatment. Genetically modified mice and cell lines were used to validate the central role of the identified cytokine and explore its downstream effectors. RESULTS: In the AML patient, disruption of cardiac metabolic homeostasis was associated with heart dysfunction after DNR-based chemotherapy. In AML mice, cardiac fatty acid utilization was attenuated, resulting in cardiac dysfunction after DNR treatment, but these phenotypes were not observed in similarly treated tumour-free mice. Furthermore, tumour cell-derived interleukin (IL)-1α was identified as a primary factor leading to DNR-induced cardiac dysfunction and administration of an anti-IL-1α neutralizing antibody could improve cardiac functions in AML mice after DNR treatment. CONCLUSIONS: This study revealed that crosstalk between tumour cells and cardiomyocytes during chemotherapy could disturb cardiac energy metabolism and impair heart function. IL-1α neutralizing antibody treatment is a promising strategy for alleviating chemotherapy-induced cardiotoxicity in AML patients.

Construction of Switch Modules for CAR-T Cell Treatment Using a Site-Specific Conjugation System.

Chimeric antigen receptor T-cell (CAR-T cell) therapy has become a promising treatment option for B-cell hematological tumors. However, few optional target antigens and disease relapse due to loss of target antigens limit the broad clinical applicability of CAR-T cells. Here, we conjugated an antibody (Ab) fusion protein, consisting of an Ab domain and a SpyCatcher domain, with the FITC-SpyTag (FITC-ST) peptide to form a bispecific safety switch module using a site-specific conjugation system. We applied the safety switch module to target CD19, PDL1, or Her2-expressing tumor cells by constructing FMC63 (anti-CD19), antiPDL1, or ZHER (anti-Her2)-FITC-ST, respectively. Those switch modules significantly improved the cytotoxic effects of anti-FITC CAR-T cells on tumor cells. Additionally, we obtained the purified CD8+ T cells by optimizing a shorter version of the CD8-binding aptamer to generate anti-FITC CD8-CAR-T cells, which combined with the CD4-FITC-ST switch module (anti-CD4) to eliminate the CD4-positive tumor cells in vitro and in vivo. Overall, we established a novel safety switch module by site-specific conjugation to enhance the antitumor function of universal CAR-T cells, thereby expanding the application scope of CAR-T therapy and improving its safety and efficacy.

Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment.

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from the immune killing of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19- lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19- lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR- T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells.

A novel aptamer beacon for rapid screening of recombinant cells and in vivo monitoring of recombinant proteins.

Recombinant protein drugs, which are typically produced by mammalian host cells, have been approved for the treatment of a range of diseases. Accordingly, systems for selecting recombinant cell lines with efficient protein expression and for testing the content of recombinant proteins in vivo are crucial to the large-scale production and application of protein-based therapeutic drugs. In this study, we designed three aptamer beacons to detect His-tag, a common label of recombinant proteins. We found that all three beacons could specifically and quantitatively measure the His-tagged recombinant proteins with a short reaction time. Among these three beacons, the 6H5-MU beacon had the highest sensitivity for His polypeptides with a detection limit of 250 ng/mL and the shortest detection time within 1 min. Furthermore, we established a rapid and highly effective recombinant cell line construction system, which could obtain monoclonal cell lines with high yields of target proteins within 21 days, by combining 6H5-MU with pSB, a novel plasmid composed of a Sleeping Beauty transposase and a transposon. Finally, 6H5-MU also discriminately tested the serum concentration of His-tagged recombinant proteins in vivo, with consistent results compared to enzyme-linked immunosorbent assay (ELISA). We thus established a rapid and high-throughput method for generating recombinant cell lines and in vivo monitoring of recombinant protein levels, thereby providing a new platform for the development and preparation of recombinant protein drugs. KEY POINTS: • The 6H5-MU aptamer beacon rapidly and accurately binds to His-tagged recombinant proteins. • A system for rapid and high-throughput generation of recombinant cell lines is established using 6H5-MU and pSB. • 6H5-MU allows in vivo monitoring of recombinant protein levels.

Dysfunctional bone marrow endothelial progenitor cells are involved in patients with myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective haematopoiesis and immune deregulation. Emerging evidence has shown the effect of bone marrow (BM) endothelial progenitor cells (EPCs) in regulating haematopoiesis and immune balance. However, the number and functions of BM EPCs in patients with different stages of MDS remain largely unknown. METHODS: Patients with MDS (N = 30), de novo acute myeloid leukaemia (AML) (N = 15), and healthy donors (HDs) (N = 15) were enrolled. MDS patients were divided into lower-risk MDS (N = 15) and higher-risk MDS (N = 15) groups according to the dichotomization of the Revised International Prognostic Scoring System. Flow cytometry was performed to analyse the number of BM EPCs. Tube formation and migration assays were performed to evaluate the functions of BM EPCs. In order to assess the gene expression profiles of BM EPCs, RNA sequencing (RNA-seq) were performed. BM EPC supporting abilities of haematopoietic stem cells (HSCs), leukaemia cells and T cells were assessed by in vitro coculture experiments. RESULTS: Increased but dysfunctional BM EPCs were found in MDS patients compared with HDs, especially in patients with higher-risk MDS. RNA-seq indicated the progressive change and differences of haematopoiesis- and immune-related pathways and genes in MDS BM EPCs. In vitro coculture experiments verified that BM EPCs from HDs, lower-risk MDS, and higher-risk MDS to AML exhibited a progressively decreased ability to support HSCs, manifested as elevated apoptosis rates and intracellular reactive oxygen species (ROS) levels and decreased colony-forming unit plating efficiencies of HSCs. Moreover, BM EPCs from higher-risk MDS patients demonstrated an increased ability to support leukaemia cells, characterized by increased proliferation, leukaemia colony-forming unit plating efficiencies, decreased apoptosis rates and apoptosis-related genes. Furthermore, BM EPCs induced T cell differentiation towards more immune-tolerant cells in higher-risk MDS patients in vitro. In addition, the levels of intracellular ROS and the apoptosis ratios were increased in BM EPCs from MDS patients, especially in higher-risk MDS patients, which may be therapeutic candidates for MDS patients. CONCLUSION: Our results suggest that dysfunctional BM EPCs are involved in MDS patients, which indicates that improving haematopoiesis supporting ability and immuneregulation ability of BM EPCs may represent a promising therapeutic approach for MDS patients.

Histamine deficiency facilitates coronary microthrombosis after myocardial infarction by increasing neutrophil-platelet interactions.

Neutrophil-platelet interactions are responsible for thrombosis as well as inflammatory responses following acute myocardial infarction (AMI). While histamine has been shown to play a crucial role in many physiological and pathological processes, its effects on neutrophil-platelet interactions in thromboinflammatory complications of AMI remain elusive. In this study, we show a previously unknown mechanism by which neutrophil-derived histamine protects the infarcted heart from excessive neutrophil-platelet interactions and redundant arterial thrombosis. Using histamine-deficient (histidine decarboxylase knockout, HDC-/- ) and wild-type murine AMI models, we demonstrate that histamine deficiency increases the number of microthrombosis after AMI, in accordance with depressed cardiac function. Histamine-producing myeloid cells, mainly Ly6G+ neutrophils, directly participate in arteriole thrombosis. Histamine deficiency elevates platelet activation and aggregation by enhancing Akt phosphorylation and leads to dysfunctional characteristics in neutrophils which was confirmed by high levels of reactive oxygen species production and CD11b expression. Furthermore, HDC-/- platelets were shown to elicit neutrophil extracellular nucleosomes release, provoke neutrophil-platelet interactions and promote HDC-expressing neutrophils recruitment in arteriole thrombosis in vivo. In conclusion, we provide evidence that histamine deficiency promotes coronary microthrombosis and deteriorates cardiac function post-AMI, which is associated with the enhanced platelets/neutrophils function and neutrophil-platelet interactions.

All-trans-retinoic acid shifts Th1 towards Th2 cell differentiation by targeting NFAT1 signalling to ameliorate immune-mediated aplastic anaemia.

Severe acquired aplastic anaemia (AA) is a serious disease characterised by autoreactive T cells attacking haematopoietic stem cells, leading to marrow hypoplasia and pancytopenia. Immunosuppressive therapy combined with antithymocyte globulin and ciclosporin can rescue most patients with AA. However, the relapse after ciclosporin withdrawal and the severe side effects of long-term ciclosporin administration remain unresolved. As such, new strategies should be developed to supplement current therapeutics and treat AA. In this study, the possibility of all-trans-retinoic acid (ATRA) as an alternative AA treatment was tested by using an immune-mediated mouse model of AA. Results revealed that ATRA inhibited T-cell proliferation, activation and effector function. It also restrained the Fas/Fasl pathway, shifted Th1 towards Th2 cell development, rebalanced T-cell subsets at a relatively high level and corrected the Th1/Th2 ratio by targeting NFAT1 signalling. In addition, ATRA inhibited Th17 cell differentiation and promoted regulatory T-cell development. Therefore, ATRA was an effective agent to improve AA treatment outcomes.

Bromodomains and Extra-Terminal (BET) Inhibitor JQ1 Suppresses Proliferation of Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolysis.

BACKGROUND Acute lymphocytic leukemia (ALL) is a common blood cancer which induces high mortality in children. Bromodomains and extra-terminal (BET) protein inhibitors, such as JQ1 and ARV-825, are promising cancer therapeutic agents that can be used by targeting c-Myc. A recent work reported that JQ1 effectively attenuates ALL in vitro by suppressing cell proliferation and accelerating apoptosis. The purpose of this research was to probe into the potential mechanism of how JQ1 inhibits ALL cell proliferation in vitro. MATERIAL AND METHODS Cell viability of ALL cells were measured by CTG after treatment by JQ1. Cell cycle analysis was done by EdU and PI staining. Cell apoptosis was assessed by Annexin V/PI staining. Glycolysis was detected using Seahorse and LC-MS kits. The expression of glycolytic rate-limiting enzymes was assessed by RNA-seq, qRT-PCR, and Western blot. RESULTS JQ1 suppressed cell proliferation by arresting the cell cycle and inducing the apoptosis of acute lymphocytic leukemia cells. JQ1 inhibited cell proliferation of B-ALL cells by restraining glycolysis. Conversely, the cell cycle block of B-ALL cells induced by JQ1 was partially abolished after pretreatment with 2-Deoxy-D-glucose (2-DG), an inhibitor of glycolysis. Furthermore, JQ1 restrained the glycolysis of B-ALL cell lines by remarkably downregulating the rate-limiting enzymes of glycolysis, such as hexokinase 2, phosphofructokinase, and lactate dehydrogenase A. Moreover, the cell cycle arrest was reversed in B-ALL cells with overexpressed c-Myc treated by JQ1, which is involved in the enhancement of glycolysis. CONCLUSIONS The BET inhibitor JQ1 suppresses the proliferation of ALL by inhibiting c-Myc-mediated glycolysis, thus providing a new strategy for the treatment of ALL.

Rapamycin ameliorates immune-mediated aplastic anemia by inhibiting the proliferation and metabolism of T cells.

Aplastic anemia (AA) is a serious blood system disease that threatens human health. At present, the main cause of this disease is believed to be immune hyperfunction. However, the specific metabolic mode involved in the occurrence of lymphocytes in AA is still unknown. In addition, whether rapamycin, a specific blocker of the mTOR signaling pathway, plays a therapeutic role by inhibiting lymphocyte metabolism remains unclear. We induced an AA mouse model through the classical immune-mediated pathway and simultaneously administered rapamycin intervention therapy. First, the AA-associated phenotypic changes and the efficacy of rapamycin in the treatment of AA were discussed. Second, the proliferation and metabolic pathway of bone marrow (BM) lymphocytes in AA and the effect of rapamycin on this process were determined. Finally, the expression levels of mTOR pathway-related proteins were analyzed. By inhibiting the mTOR signaling pathway, rapamycin could ameliorate the phenotype of the immune-mediated AA model and inhibit the proliferation of T cells by preventing cell cycle transition from G0 to G1 phase. Moreover, we found that mitochondrial oxidative phosphorylation is involved in the metabolic reprogramming of T cells in AA and that rapamycin can inhibit this process. We confirmed that mitochondrial oxidative phosphorylation is involved in the metabolic reprogramming of T cells in AA and further extended the mechanism of rapamycin in treating AA by inhibiting the mTOR signaling pathway. This viewpoint may provide a new therapeutic idea for clinical applications.

LGR6 promotes osteogenesis by activating the Wnt/ß-catenin signaling pathway.

Leucine-rich repeat containing G-protein-coupled receptor 6 (LGR6) is a member of the rhodopsin-like 7-transmembrane domain receptor superfamily and has high homology to LGR4 and LGR5. LGR6 is highly expressed in osteoblastic progenitors, and LGR6-deficient mice show nail and bone regeneration defect. However, the effect of LGR6 on the osteogenic differentiation of osteoblastic progenitors and its underlying mechanisms are largely unknown. In this study, we overexpressed and knockdown LGR6 with lentivirus in the preosteoblastic cell MC3T3-E1 to observe the effect of LGR6 on osteogenic differentiation and explore its possible molecular mechanism. LGR6 overexpression promoted osteogenic differentiation and mineralization by stabilizing ß-catenin to potentiate the Wnt/ß-catenin signaling pathway in MC3T3-E1 cells. Conversely, LGR6 knockdown inhibited osteogenic differentiation and mineralization by enhancing ß-catenin degradation to inactivate the Wnt/ß-catenin signaling pathway. These results reveal that LGR6 is highly expressed in osteoblastic progenitors, and promotes osteogenesis by enhancing ß-catenin stability to strengthen the Wnt signaling pathway. This study provides an important reference into the exact mechanisms of osteogenic differentiation.

Impairment of bone marrow endothelial progenitor cells in acute graft-versus-host disease patients after allotransplant.

Graft-versus-host disease (GVHD) is a major complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT) that is frequently associated with bone marrow (BM) suppression, and clinical management is challenging. BM endothelial progenitor cells (EPCs) play crucial roles in the regulation of haematopoiesis and thrombopoiesis. However, little is known regarding the functional roles of BM EPCs in acute GVHD (aGVHD) patients. In the current prospective case-control study, reduced and dysfunctional BM EPCs, characterized by decreased migration and angiogenesis capacities and increased levels of reactive oxygen species (ROS) and apoptosis, were found in aGVHD patients compared with those without aGVHD. Moreover, lower frequency and increased levels of ROS, apoptosis and DNA damage, but reduced colony-forming unit-plating efficiency were found in BM CD34+ cells of aGVHD patients compared with those without aGVHD. The severity of aGVHD and GVHD-mediated cytopenia was associated with BM EPC impairment in aGVHD patients. In addition, the EPC impairment positively correlated with ROS level. Taken together, our results suggest that reduced and dysfunctional BM EPCs may be involved in the pathogenesis of aGVHD. Although these findings require validation, our data indicate that improvement of BM EPCs may represent a promising therapeutic approach for aGVHD patients.

An unbalanced monocyte macrophage polarization in the bone marrow microenvironment of patients with poor graft function after allogeneic haematopoietic stem cell transplantation.

Poor graft function (PGF) is a severe complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). Murine studies have demonstrated that effective haematopoiesis depends on the specific bone marrow (BM) microenvironment. Increasing evidence shows that BM macrophages (MФs), which constitute an important component of BM immune microenvironment, are indispensable for the regulation of haematopoietic stem cells (HSCs) in the BM. However, little is known about the number and function of BM MФs or whether they directly interact with HSCs in PGF patients. In the current prospective case-control study, PGF patients showed a significant increase in classically activated inflammatory MФs (M1; 2·18 ± 0·11% vs. 0·82 ± 0·06%, P < 0·0001), a striking reduction in alternatively activated anti-inflammatory MФs (M2; 3·02 ± 0·31% vs. 21·89 ± 0·90%, P < 0·0001), resulting in a markedly increased M1/M2 ratio (0·82 ± 0·06 vs. 0·06 ± 0·002; P < 0·0001) in the BM compared with good graft function patients. Meanwhile, standard monocyte subsets were altered in PGF patients. Dysfunctional BM MФs, which were characterized by reduced proliferation, migration and phagocytosis, were evident in PGF patients. Furthermore, BM MФs from PGF patients with high tumour necrosis factor-α and interleukin 12 levels and low transforming growth factor-ß levels, led to impaired BM CD34+ cell function. In summary, our data indicate that an unbalanced BM M1/M2 ratio and dysfunctional MФs may contribute to the occurrence of PGF following allo-HSCT.

Fev regulates hematopoietic stem cell development via ERK signaling.

Reprogramming of somatic cells to desired cell types holds great promise in regenerative medicine. However, production of transplantable hematopoietic stem cells (HSCs) in vitro by defined factors has not yet been achieved. Therefore, it is critical to fully understand the molecular mechanisms of HSC development in vivo. Here, we show that Fev, an ETS transcription factor, is a pivotal regulator of HSC development in vertebrates. In fev-deficient zebrafish embryos, the first definitive HSC population was compromised and fewer T cells were found in the thymus. Genetic and chemical analyses support a mechanism whereby Fev regulates HSC through direct regulation of ERK signaling. Blastula transplant assay demonstrates that Fev regulation of HSC development is cell autonomous. Experiments performed with purified cord blood show that fev is expressed and functions in primitive HSCs in humans, indicating its conserved role in higher vertebrates. Our data indicate that Fev-ERK signaling is essential for hemogenic endothelium-based HSC development.

Construction of a versatile fusion protein for targeted therapy and immunotherapy.

Antibody (Ab)-based drugs have been widely used in targeted therapies and immunotherapies, leading to significant improvements in tumor therapy. However, the failure of Ab therapy due to the loss of target antigens or Ab modifications that affect its function limits its application. In this study, we expanded the application of antibodies (Abs) by constructing a fusion protein as a versatile tool for Ab-based target cell detection, delivery, and therapy. We first constructed a SpaC Catcher (SpaCC for short) fusion protein that included the C domains of Staphylococcal protein A (SpaC) and the SpyCatcher. SpaCC conjugated with SpyTag-X (S-X) to form the SpaCC-S-X complex, which binds non-covalently to an Ab to form the Ab-SpaCC-S-X protein complex. The "X" can be a variety of small molecules such as fluoresceins, cell-penetrating peptide TAT, Monomethyl auristatin E (MMAE), and DNA. We found that Ab-SpaCC-S-FITC(-TAT) could be used for target cell detection and delivery. Besides, we synthesized the Ab-SpaCC-SN3-MMAE complex by linking Ab with MMAE by SpaCC, which improved the cytotoxicity of small molecule toxins. Moreover, we constructed an Ab-DNA complex by conjugating SpaCC with the aptamer (Ap) and found that Ab-SpaCC-SN3-Ap boosted the tumor-killing function of T-cells by retargeting tumor cells. Thus, we developed a multifunctional tool that could be used for targeted therapies and immunotherapies, providing a cheap and convenient novel drug development strategy.

Interleukin-27 Promotes the Generation of Myeloid-derived Suppressor Cells to Alleviate Graft-versus-host Disease.

BACKGROUND: Stimulation of myeloid-derived suppressor cell (MDSC) formation represents a potential curative therapeutic approach for graft-versus-host disease (GVHD), which significantly impacts the prognosis of allogeneic hematopoietic stem cell transplantation. However, the lack of an effective strategy for inducing MDSC production in vivo has hindered their clinical application. In our previous study, MDSC expansion was observed in interleukin (IL)-27-treated mice. METHODS: In this study, we overexpressed exogenous IL-27 in mice using a recombinant adeno-associated virus vector to investigate its therapeutic and exacerbating effects in murine GVHD models. RESULTS: In our study, we demonstrated that exogenous administration of IL-27 significantly suppressed GVHD development in a mouse model. We found that IL-27 treatment indirectly inhibited the proliferation and activation of donor T cells by rapidly expanding recipient and donor myeloid cells, which act as MDSCs after irradiation or under inflammatory conditions, rather than through regulatory T-cell expansion. Additionally, IL-27 stimulated MDSC expansion by enhancing granulocyte-monocyte progenitor generation. Notably, we verified that IL-27 signaling in donor T cells exerted an antagonistic effect on GVHD prevention and treatment. Further investigation revealed that combination therapy involving IL-27 and T-cell depletion exhibited remarkable preventive effects on GVHD in both mouse and xenogeneic GVHD models. CONCLUSIONS: Collectively, these findings suggest that IL-27 promotes MDSC generation to reduce the incidence of GVHD, whereas targeted activation of IL-27 signaling in myeloid progenitors or its combination with T-cell depletion represents a potential strategy for GVHD therapy.

CAR-Aptamers Enable Traceless Enrichment and Monitoring of CAR-Positive Cells and Overcome Tumor Immune Escape.

Chimeric antigen receptor (CAR)-positive cell therapy, specifically with anti-CD19 CAR-T (CAR19-T) cells, achieves a high complete response during tumor treatment for hematological malignancies. Large-scale production and application of CAR-T therapy can be achieved by developing efficient and low-cost enrichment methods for CAR-T cells, expansion monitoring in vivo, and overcoming tumor escape. Here, novel CAR-specific binding aptamers (CAR-ap) to traceless sort CAR-positive cells and obtain a high positive rate of CAR19-T cells is identified. Additionally, CAR-ap-enriched CAR19-T cells exhibit similar antitumor capacity as CAR-ab (anti-CAR antibody)-enriched CAR-T cells. Moreover, CAR-ap accurately monitors the expansion of CAR19-T cells in vivo and predicts the prognosis of CAR-T treatment. Essentially, a novel class of stable CAR-ap-based bispecific circular aptamers (CAR-bc-ap) is constructed by linking CAR-ap with a tumor surface antigen (TSA): protein tyrosine kinase 7 (PTK7) binding aptamer Sgc8. These CAR-bc-aps significantly enhance antitumor cytotoxicity with a loss of target antigens by retargeting CAR-T cells to the tumor in vitro and in vivo. Overall, novel CAR-aptamers are screened for traceless enrichment, monitoring of CAR-positive cells, and overcoming tumor cell immune escape. This provides a low-cost and high-throughput approach for CAR-positive cell-based immunotherapy.

Application of a Novel Aptamer Beacon for Rapid Detection of IgG1 Antibody Drugs.

Antibody drugs have been widely used to treat many diseases and are the fastest-growing drug class. IgG1 is the most common type of antibody because of its good serum stability; however, effective methods for the rapid detection of IgG1-type antibodies are lacking. In this study, we designed two aptamer molecules derived from the reported aptamer probe that has been proven to bind to the Fc fragment of the IgG1 antibody. The results showed that Fc-1S could specifically bind to the human IgG1 Fc proteins. In addition, we modified the structure of Fc-1S and constructed three aptamer molecular beacons that could quantitatively detect IgG1-type antibodies within a short time. Furthermore, we unveiled that the Fc-1S37R beacon has the highest sensitivity for IgG1-type antibodies with a detection limit of 48.82813 ng/mL and can accurately detect serum antibody concentrations in vivo with consistent results to ELISA. Therefore, Fc-1S37R is an efficient method for the production monitoring and quality control of IgG1-type antibodies to enable the large-scale production and application of antibody drugs.

A novel His-tag-binding aptamer for recombinant protein detection and T cell-based immunotherapy.

Screening novel aptamers for recombinant protein detection is of great significance in industrial mass production of antibody drugs. In addition, construction of structurally stable bispecific circular aptamers (bc-apts) may provide a tumor-targeted treatment strategy by simultaneously binding two different cell types. In this study, we obtained a high-affinity hexahistidine tag (His-tag)-binding aptamer 20S and explored its application in recombinant protein detection and T cell-based immunotherapy. We developed a new molecular beacon (MB) 20S-MB to detect His-tagged proteins in vitro and in vivo with high sensitivity and specificity, and the results showed high consistency with the enzyme-linked immunosorbent assay (ELISA). Moreover, we constructed two kinds of bc-apts by cyclizing 20S or another His-tag-binding aptamer, 6H5-MU, with Sgc8, which specifically recognizes protein tyrosine kinase 7 (PTK7) on tumor cells. After forming a complex with His-tagged OKT3, an anti-CD3 antibody for T cell activation, we utilized these aptamer-antibody complexes (ap-ab complex) to enhance cytotoxicity of T cells by linking T cells and target cells together, and 20S-sgc8 exhibited antitumor efficacy superior to that of 6H5-sgc8. In conclusion, we screened a novel His-tag-binding aptamer and used it to construct a new type of MB for rapid detection of recombinant proteins, as well as establish a feasible approach for T cell-based immunotherapy.

Targeting DNA polymerase ß elicits synthetic lethality with mismatch repair deficiency in acute lymphoblastic leukemia.

Mismatch repair (MMR) deficiency has been linked to thiopurine resistance and hypermutation in relapsed acute lymphoblastic leukemia (ALL). However, the repair mechanism of thiopurine-induced DNA damage in the absence of MMR remains unclear. Here, we provide evidence that DNA polymerase ß (POLB) of base excision repair (BER) pathway plays a critical role in the survival and thiopurine resistance of MMR-deficient ALL cells. In these aggressive resistant ALL cells, POLB depletion and its inhibitor oleanolic acid (OA) treatment result in synthetic lethality with MMR deficiency through increased cellular apurinic/apyrimidinic (AP) sites, DNA strand breaks and apoptosis. POLB depletion increases thiopurine sensitivities of resistant cells, and OA synergizes with thiopurine to kill these cells in ALL cell lines, patient-derived xenograft (PDX) cells and xenograft mouse models. Our findings suggest BER and POLB's roles in the process of repairing thiopurine-induced DNA damage in MMR-deficient ALL cells, and implicate their potentials as therapeutic targets against aggressive ALL progression.