Functionality of a bicistronic construction containing HEXA and HEXB genes encoding β-hexosaminidase A for cell-mediated therapy of GM2 gangliosidoses

Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency ofβ-hexosaminidase A(HexA)enzyme,which results in the accumulation of GM2 gangliosides in the nervous system cells.In this work,we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexAα-andβ-subunit genes separated by the nucleotide sequence of a P2A peptide(HEXA-HEXB).The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells(hUCBMCs).Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times,respectively,compared with the conditioned medium of native cells.Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins,and an uncleaved protein containing HEXA+HEXB linked by the P2A peptide.Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out,and the HexA enzymatic activity in the blood plasma of experimental animals,as well as the number of live cells of immune system organs(spleen,thymus,bone marrow,lymph nodes)were determined.A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9(by 2.5 and 3 times,respectively)after the administration of hUCBMCsHEXA-HEXB was shown.At the same time,the number of live cells in the studied organs remained unchanged.Thus,the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo.We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers,such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses.Based on the obtained data,it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses.The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University(No.23)on June 30,2020.

Evaluation of antibody-dependent cell-mediatedcy totoxicity activity and cetuximab response in KRAS wildtype metastatic colorectal cancer patients

AIM:To investigate the prognostic role of invariant natural killer T(iNKT) cells and antibody-dependent cell-mediated cytotoxicity(ADCC) in wild type KRAS metastatic colorectal cancer(mC RC) patients treated with cetuximab.METHODS: Forty-one KRAS wt mC RC patients,treated with cetuximab and irinotecan-based chemotherapy in Ⅱ and Ⅲ lines were analyzed. Genotyping of single nucleotide polymorphism(SNP)s in the FCGR2A,FCGR3A and in the 3' untranslated regions of KRAS and mutational analysis for KRAS,BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprepperipheral blood mononuclear cell and iN KT cells were defined by co-expression of CD3,TCRVα24,TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity,measuring lactate dehydrogenase release.RESULTS: At basal,mCRC patients performing ADCC activity above the median level(71%) showed an improved overall survival(OS) compared to patients with ADCC below(median 16 vs 8 mo;P=0.026). We did not find any significant correlation of iN KT cells with OS(P=0.19),albeit we observed a trend to a longer survival after 10 mo in patients with iN KT above median basal level(0.382 cells/microliter). Correlation of OS and progression-free survival(PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2 A and TT in FCGR3A presented a trend of longer PFS(median 9 vs 5 mo;P=0.064). Chemotherapy impacted both iN KT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment.CONCLUSION: Our results suggest a link between iN KT cells,basal ADCC activity,genotypes in FCGR2A and FCGR3A,and efficacy of cetuximab in KRAS wt mC RC patients.

Histopathological analysis of infiltrating T cell subsets in acute T cell-mediated rejection in the kidney transplant

AIM To compare the differential immune T cell subset com-position in patients with acute T cell-mediated rejection in the kidney transplant with subset composition in the absence of rejection, and to explore the association of their respective immune profiles with kidney transplant outcomes.METHODS A pilot cross-sectional histopathological analysis of the immune infiltrate was performed using immunohistochemistry in a cohort of 14 patients with acute T cellmediated rejection in the kidney transplant and 7 kidney transplant patients with no rejection subjected to biopsy to investigate acute kidney transplant dysfunction. All patients were recruited consecutively from 2012 to 2014 at the Singapore General Hospital. Association of the immune infiltrates with kidney transplant outcomes at up to 54 mo of follow up was also explored prospectively.RESULTS In a comparison to the absence of rejection, acute T cell-mediated rejection in the kidney transplant was characterised by numerical dominance of cytotoxic T lymphocytes over Foxp3^+ regulatory T cells, but did not reach statistical significance owing to the small sample size in our pilot study. There was no obvious difference in absolute numbers of infiltrating cytotoxic T lymphocytes, Foxp3^+ regulatory T cells and Th17 cells between the two patient groups when quantified separately. Our exploratory analysis on associations of T cell subset quantifications with kidney transplant outcomes revealed that the degree of Th17 cell infiltration was significantly associated with shorter time to doubling of creatinine and shorter time to transplant loss.CONCLUSION Although this was a small pilot study, results support our suspicion that in kidney transplant patients the immune balance in acute T cell-mediated rejection is tilted towards the pro-rejection forces and prompt larger and more sophisticated studies.

Increasingβ-hexosaminidase A activity using genetically modified mesenchymal stem cells

GM2 gangliosidoses are a group of autosomal-recessive lysosomal storage disorde rs.These diseases result from a deficiency of lysosomal enzymeβ-hexosaminidase A(HexA),which is responsible for GM2 ganglioside degradation.HexA deficiency causes the accumulation of GM2-gangliosides mainly in the nervous system cells,leading to severe progressive neurodegeneration and neuroinflammation.To date,there is no treatment for these diseases.Cell-mediated gene therapy is considered a promising treatment for GM2 gangliosidoses.This study aimed to evaluate the ability of genetically modified mesenchymal stem cells(MSCs-HEXA-HEXB)to restore HexA deficiency in Tay-Sachs disease patient cells,as well as to analyze the functionality and biodistribution of MSCs in vivo.The effectiveness of HexA deficiency cross-correction was shown in mutant MSCs upon intera ction with MSCs-HEXA-HEXB.The results also showed that the MSCs-HEXA-HEXB express the functionally active HexA enzyme,detectable in vivo,and intravenous injection of the cells does not cause an immune response in animals.These data suggest that genetically modified mesenchymal stem cells have the potentials to treat GM2 gangliosidoses.

Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1^(high)CD11b^(+)cells elimination in hepatocellular carcinoma

Cryoablation(CRA)and microwave ablation(MWA)are two main local treatments for hepatocellular carcinoma(HCC).However,which one is more curative and suitable for combining with immunotherapy is still controversial.Herein,CRA induced higher tumoral PD-L1 expression and more T cells infiltration,but less PD-L1^(high)CD11b^(+)myeloid cells infiltration than MWA in HCC.Furthermore,CRA had better curative effect than MWA for anti-PD-L1 combination therapy in mouse models.Mechanistically,anti-PD-L1 antibody facilitated infiltration of CD8^(+)T cells by enhancing the secretion of CXCL9 from cDC1 cells after CRA therapy.On the other hand,anti-PD-L1 antibody promoted the infiltration of NK cells to eliminate PD-L1^(high)CD11b^(+)myeloid cells by antibody-dependent cell-mediated cytotoxicity(ADCC)effect after CRA therapy.Both aspects relieved the immunosuppressive microenvironment after CRA therapy.Notably,the wild-type PD-L1 Avelumab(Bavencio),compared to the mutant PD-L1 atezolizumab(Tecentriq),was better at inducing the ADCC effect to target PD-L1^(high)CD11b^(+)myeloid cells.Collectively,our study uncovered the novel insights that CRA showed superior curative effect than MWA in combining with anti-PD-L1 antibody by strengthening CTL/NK cell immune responses,which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC.

Secondary pulmonary infection by Fusarium solani and Aspergillus niger during systemic steroid treatment for COVID-19:A case report

BACKGROUND Coronavirus disease 2019(COVID-19)-associated invasive pulmonary aspergillosis presents a diagnostic challenge due to its non-specific clinical/imaging features,as well as the fact that the proposed clinically diagnostic algorithms do not necessarily apply to COVID-19 patients.In addition,Fusarium spp.is a rare cause of opportunistic life-threatening fungal infections.Disseminated Fusarium infection in an immunocompromised host is intractable,with a high likelihood of resulting mortality.To our knowledge,this is the first case of secondary pulmonary infection by Fusarium solani(F.solani)and Aspergillus niger(A.niger)during systemic steroid treatment for COVID-19.CASE SUMMARY A 62-year-old male was transported to our hospital by ambulance with a complaint of fever and dyspnea.We established a diagnosis of pneumococcal pneumonia,complicated with COVID-19 and septic shock,together with acute renal failure.He was admitted to the intensive care unit,to be treated with piperacillin/tazobactam,vancomycin,and 6.6 mg per day of dexamethasone sodium phosphate,along with noradrenaline as a vasopressor,ventilator management,and continuous hemodiafiltration.His condition improved,and we finished the vasopressor on the fifth hospital day.We administered dexamethasone for ten days,and finished the course of treatment.On the eleventh day,patient respiratory deterioration was observed,and a computed tomography scan showed an exacerbation of bilateral ground-glass-opacity-like consolidation,together with newly appeared cavitary lesions in the lung.we changed antibiotics to meropenem plus vancomycin.In addition,a fungal infection was considered as a possibility based on microscopic findings of sputum,and we began coadministration of voriconazole.However,the pneumonia worsened,and the patient died on the seventeenth day of illness.Later,F.solani and A.niger were identified from sputum collected on the twelfth day.It was believed that he developed a cell-mediated immune deficiency during COVID-19 treatment,which led to the complication of pneumonia caused by the above-mentioned fungi,contributing to his death.CONCLUSION Because early initiation of intense antifungal therapy offers the best chance for survival in pulmonary fusariosis,computed tomography scans and appropriate microbiologic investigations should be obtained for severely immunocompromised patients.

Alpha-fetoprotein specific CD4 and CD8 T cell responses in patients with hepatocellular carcinoma

The presence of CD8 T cell responses to tumor associated antigens have been reported in patients with different malignancies. However, there is very little inf ormation on a comparable CD8 and CD4 T cell response to a tumor antigen in liver cancer patients. Here, we re-examine the kinetic and the pattern of T helper 1 and cytotoxic T lymphocyte responses to alpha-fetoprotein (AFP),a tumor rejection antigen in hepatocellular carcinoma (HCC). Then, we discuss the possibility of using AFP-based immunotherapy in combination with necrotizing treatments in HCC patients.

Current status of cell-mediated regenerative therapies for human spinal cord injury

During the past decade, significant advances have been made in refinements for regenerative therapies following human spinal cord injury （SCI）. Positive results have been achieved with different types of cells in various clinical studies of SCI. In this review, we summarize recently-completed clinical trials using cell- mediated regenerative therapies for human SCI, together with ongoing trials using neural stem cells. Specifically, clinical studies published in Chinese journals are included. These studies show that current transplantation therapies are relatively safe, and have provided varying degrees of neurological recovery. However, many obstacles exist, hindering the introduction of a specific clinical therapy, including complications and their causes, selection of the target population, and optimization of transplantation material. Despite these and other challenges, with the collaboration of research groups and strong support from various organizations, cell-mediated regenerative therapies will open new perspectives for SCI treatment.

Extracellular vesicles in the diagnosis and treatment of central nervous system diseases

Extracellular vesicles,including exosomes and microvesicles,play a fundamental role in the activity of the nervous system,participating in signal transmission between neurons and providing the interaction of central nervous system with all body systems.In many neurodegenerative diseases,neurons pack toxic substances into vesicles and release them into the extracellular space,which leads to the spread of misfolded neurotoxic proteins.The contents of neuron-derived extracellular vesicles may indicate pathological changes in the central nervous system,and the analysis of extracellular vesicle molecular content contributes to the development of non-invasive methods for the diagnosis of many central nervous system diseases.Extracellular vesicles of neuronal origin can be isolated from various biological fluids due to their ability to cross the blood-brain barrier.Today,the diagnostic potential of almost all toxic proteins involved in nervous system disease pathogenesis,specificallyα-synuclein,tau protein,superoxide dismutase 1,FUS,leucine-rich repeat kinase 2,as well as some synaptic proteins,has been well evidenced.Special attention is paid to extracellular RNAs mostly associated with extracellular vesicles,which are important in the onset and development of many neurodegenerative diseases.Depending on parental cell type,extracellular vesicles may have different therapeutic properties,including neuroprotective,regenerative,and anti-inflammatory.Due to nano size,biosafety,ability to cross the blood-brain barrier,possibility of targeted delivery and the lack of an immune response,extracellular vesicles are a promising vehicle for the delivery of therapeutic substances for the treatment of neurodegenerative diseases and drug delivery to the brain.This review describes modern approaches of diagnosis and treatment of central nervous system diseases using extracellular vesicles.

Acute Morphine Administration Reduces Cell-Mediated Immunity and Induces Reactivation of Latent Herpes Simplex Virus Type 1 in BALB/c Mice

Acute morphine administration is known to alter the course of herpes simplex virus infection. In this study, the effect of acute morphine administration on the reactivation of latent herpes was investigated in a mouse model. Because of the important role of cytolytic T lymphocyte （CTL） activity in the inhibition of herpes simplex virus type 1 （HSV-1） reactivation, the effect of acute morphine administration on CTL responses was also evaluated. Furthermore, lymphocyte proliferation and IFN-γ production were evaluated for their roles in the induction of the CTL response. The findings showed that acute morphine administration significantly reduced CTL responses, lymphocyte proliferation, and IFN-γ production. Furthermore, acute morphine administration has been shown to reactivate latent HSV-γ. Previous studies have shown that cellular immune responses have important roles in the inhibition of HSV reactivation. These findings suggest that suppression of a portion of the cellular immune response after acute morphine administration may constitute one part of the mechanism that induces HSV reactivation.

Chronic rejection after liver transplantation:Opening the Pandora’s box

Chronic rejection(CR)of liver allografts causes damage to intrahepatic vessels and bile ducts and may lead to graft failure after liver transplantation.Although its prevalence has declined steadily with the introduction of potent immunosuppressive therapy,CR still represents an important cause of graft injury,which might be irreversible,leading to graft loss requiring re-transplantation.To date,we still do not fully appreciate the mechanisms underlying this process.In addition to T cell-mediated CR,which was initially the only recognized type of CR,recently a new form of liver allograft CR,antibody-mediated CR,has been identified.This has indeed opened an era of thriving research and renewed interest in the field.Liver biopsy is needed for a definitive diagnosis of CR,but current research is aiming to identify new non-invasive tools for predicting patients at risk for CR after liver transplantation.Moreover,the minimization or withdrawal of immunosuppressive therapy might influence the establishment of subclinical CR-related injury,which should not be disregarded.Therapies for CR may only be effective in the“early”phases,and a tailored management of the immunosuppression regimen is essential for preventing irreversible liver damage.Herein,we provide an overview of the current knowledge and research on CR,focusing on early detection,identification of non-invasive biomarkers,immunosuppressive management,re-transplantation and future perspectives of CR.

Primary intestinal lymphangiectasia with generalized warts

Primary intestinal lymphangiectasia(PIL) is a rare protein-losing enteropathy with lymphatic leakage into the small intestine. Dilated lymphatics in the small intestinal wall and mesentery are observed in this disease. Laboratory tests of PIL patients revealed hypoalbuminemia, lymphocytopenia, hypogammaglobulinemia and increased stool α-1 antitrypsin clearance. Cell-mediated immunodeficiency is also present in PIL patients because of loss of lymphocytes. As a result, the patients are vulnerable to chronic viral infection and lymphoma. However, cases of PIL with chronic viral infection, such as human papilloma virus-induced warts, are rarely reported. We report a rare case of PIL with generalized warts in a 36-year-old male patient. PIL was diagnosed by capsule endoscopy and colonoscopic biopsy with histological tissue confirmation. Generalized warts were observed on the head, chest, abdomen, back, anus, and upper and lower extremities, including the hands and feet of the patient.

Efficient generation of the mouse model with a defined point mutation through haploid cell-mediated gene editing

Generation of mouse models carrying a defined point mutation,especially disease-related point mutations,is of considerable interest for research in biology and medicine.The standard method based on embryonic stem cell（ESC）-mediated homologous recombination（HR）is time-and labor-consuming.

Nanoparticle‑Loaded Polarized‑Macrophages for Enhanced Tumor Targeting and Cell‑Chemotherapy

Cell therapy is a promising strategy for cancer therapy.However,its therapeutic efficiency remains limited due to the complex and immunosuppressive nature of tumor microenvironments.In this study,the“cell-chemotherapy”strategy was presented to enhance antitumor efficacy.M1-type macrophages,which are therapeutic immune cells with both of immunotherapeutic ability and targeting ability,carried sorafenib(SF)-loaded lipid nanoparticles(M1/SLNPs)were developed.M1-type macrophages were used both as therapeutic tool to provide immunotherapy and as delivery vessel to target deliver SF to tumor tissues for chemotherapy simultaneously.M1-type macrophages were obtained by polarizing macrophages using lipopolysaccharide,and M1/SLNPs were obtained by incubating M1-type macrophages with SLNP.Tumor accumulation of M1/SLNP was increased compared with SLNP(p<0.01),which proved M1/SLNP could enhance tumor targeting of SF.An increased ratio of M1-type macrophages to M2-type macrophages,and the CD3^+CD4^+T cells and CD3^+CD8^+T cell quantities in tumor tissues after treatment with M1/SLNP indicated M1/SLNP could relieve the immunosuppressive tumor microenvironments.The tumor volumes in the M1/SLNP group were significantly smaller than those in the SLNP group(p<0.01),indicating M1/SLNP exhibited enhanced antitumor efficacy.Consequently,M1/SLNP showed great potential as a novel cellchemotherapeutic strategy combining both cell therapy and targeting chemotherapy.

Proteomics for rejection diagnosis in renal transplant patients: Where are we now?

Rejection is one of the key factors that determine the long-term allograft function and survival in renal transplant patients. Reliable and timely diagnosis is important to treat rejection as early as possible. Allograft biopsies are not suitable for continuous monitoring of rejection. Thus, there is an unmet need for non-invasive methods to diagnose acute and chronic rejection. Proteomics in urine and blood samples has been explored for this purpose in 29 studies conducted since 2003. This review describes the different proteomic approaches and summarizes the results from the studies that examined proteomics for the rejection diagnoses. The potential limitations and open questions in establishing proteomic markers for rejection are discussed, including ongoing trials and future challenges to this topic.

The Effect of Tremella Fuciformis Spores Polysaccharides (TSP) on Immune Cellular Function of Mice in Vitro

TSP could markedly enhance the proliferative response of the murine splenocyte to LPS and induce the mitogenesis of the spleen cells.Furthermore,it was able to augment the activity of natural killer cell and ADGG;at a dosage of 25-250μg/ml,the ability of splenocytes to produce IL-2 induced by onA had been improved; at the concentration of 250μg／ml or more,TSP could inhibit the proliferative response of the murine lymphocyte to GonA and the ~3-HTdR spontaneous incorporation rate of thymocytes,and the inhibitory action ran in paralell with the increase in concentration of TSP.

Electrophoretic Analysis on RNA of Deltamethrin－resistant Culex Pipiens Pallens

TSP could markedly enhance the proliferative response of the murine splenocyte to LPS and induce the mitogenesis of the spleen cells.Furthermore,it was able to augment the activity of natural killer cell and ADGG;at a dosage of 25-250μg/ml,the ability of splenocytes to produce IL-2 induced by onA had been improved; at the concentration of 250μg/ml or more,TSP could inhibit the proliferative response of the murine lymphocyte to GonA and the ￣3-HTdR spontaneous incorporation rate of thymocytes,and the inhibitory action ran in paralell with the increase in concentration of TSP.

The exciting potential of nanotherapy in brain-tumor targeted drug delivery approaches

Delivering therapeutics to the central nervous system（CNS） and brain-tumor has been a major challenge. The current standard treatment approaches for the brain-tumor comprise of surgical resection followed by immunotherapy, radiotherapy, and chemotherapy. However, the current treatments are limited in providing significant benefits to the patients and despite recent technological advancements; brain-tumor is still challenging to treat. Brain-tumor therapy is limited by the lack of effective and targeted strategies to deliver chemotherapeutic agents across the blood-brain barrier（BBB）. The BBB is the main obstacle that must be overcome to allow compounds to reach their targets in the brain. Recent advances have boosted the nanotherapeutic approaches in providing an attractive strategy in improving the drug delivery across the BBB and into the CNS. Compared to conventional formulations, nanoformulations offer significant ad vantages in CNS drug delivery approaches. Considering the above facts, in this review, the physiological/anatomical features of the brain-tumor and the BBB are briefly discussed. The drug transport mechanisms at the BBB are outlined. The approaches to deliver chemotherapeutic drugs across the CNS into the brain-tumor using nanocarriers are summarized. In addition, the challenges that need to be addressed in nanotherapeutic approaches for their enhanced clinical application in brain-tumor therapy are discussed.

Activin-Directed Differentiation of Human Embryonic Stem Cells Differentially Modulates Alveolar Epithelial Wound Repair via Paracrine Mechanism

Differentiated embryonic stem cells (ESC) can ameliorate lung inflammation and fibrosis in animal lung injury models;therefore, ESC, or their products, could be candidates for regenerative therapy for incurable lung diseases, such as idiopathic pulmonary fibrosis (IPF). In this study, we have investigated the paracrine effect of differentiated and undifferentiated human ESC on alveolar epithelial cell (AEC) wound repair. hESC line, SHEF-2 cells were differentiated with Activin treatment for 22 days in an embryoid body (EB) suspension culture. Conditioned media (CM) which contain cell secretory factors were collected at different time points of differentiation. CM were then tested onin vitro?wound repair model with human type II AEC line, A549 cells (AEC). Our study demonstrated that CM originated from undifferentiated hESC significantly inhibited AEC wound repair when compared to the control. Whereas, CM originated from Activin-directed hESC differentiated cell population demonstrated a differential reparative effect on AEC wound repair model. CM obtained from Day-11 of differentiation significantly enhanced AEC wound repair in comparison to CM collected from pre- and post-Day-11 of differentiation. Day-11 CM enhanced AEC wound repair through significant stimulation of cell migration and cell proliferation. RT-PCR and immunocytochemistry confirmed that Day-11 CM was originated form a mixed population of endodermal/mesodermal differentiated hESC. This report suggests a putative paracrine-mediated epithelial injury healing mechanism by hESC secreted products, which is valuable in the development of novel stem cell-based therapeutic strategies.

Novel Strategies for Redirecting NK Cells in Cancer Immunotherapy

Natural killer(NK)cells act as a first line of defense in innate immune system against new malignant transformed cells without prior exposure to tumor antigens.The activity of NK cells is tightly controlled by a balance between inhibitory receptors and activating receptors,and when activation signals outweigh inhibitory signals,NK cells can mediate a response to eliminate the targeted cells.As a bridge between the innate and adaptive immune responses to enhance destruction of tumors,NK cells kill their tumor targets through a variety of mechanisms,including receptor-mediated cytotoxicity,antibody-dependent cell-mediated cytotoxicity(ADCC),death receptor Fas/FasL signaling pathway-mediated cancer apoptosis.However,the efficacy of using NK cells for tumor immunotherapy has been limited by a lack of antigen specificity.To overcome this limitation,Vallera and colleagues developed a bispecific killer cell engager(BiKE),which is comprised of a first ScFv that recognizes a tumor antigen and a second ScFv against CD16(expressed on NK cells)to trigger ADCC.To overcome the poor expansion of NK cells in vivo,a novel trispecific killer cell engager(TriKE)was evolved from the BiKE by the same team.In this molecule,IL-15 was integrated to promote NK cell expansion,thereby eliciting superior NK cytotoxicity and NK cell persistence in vivo compared to BiKE.In order to simultaneously target drug-refractory cancer stem cells(CSC)and cancer cells,a novel tetraspecific killer engager(TetraKE)comprising anti-CD133,EpCAM,CD16 ScFvs and a sustaining IL-15 signal cross-linker was recently developed.Compared to BiKE or TriKE,TetraKE1615EpCAM133 was highly specific against EpCAM-and CD133-bearing cells,leading to enhanced NK cell proliferation,prolonged survival and a limited cytokine response.This TetraKE represents a promising new modality for immunotherapy.