MicroRNA in United Airway Diseases

The concept of united airway diseases (UAD) has received increasing attention in recent years. Sustained and increased inflammation is a common feature of UAD, which is inevitably accompanied with marked gene modification and tight gene regulation. However, gene regulation in the common inflammatory processes in UAD remains unclear. MicroRNA (miRNA), a novel regulator of gene expression, has been considered to be involved in many inflammatory diseases. Although there are an increasing number of studies of miRNAs in inflammatory upper and lower airway diseases, few miRNAs have been identified that directly link the upper and lower airways. In this article, therefore, we reviewed the relevant studies available in order to improve the understanding of the roles of miRNAs in the interaction and pathogenesis of UAD.     

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.     

Monoclonal Antibodies and Invasive Aspergillosis: Diagnostic and Therapeutic Perspectives

Invasive aspergillosis (IA) is a life-threatening fungal disease that causes high morbidity and mortality in immunosuppressed patients. Early and accurate diagnosis and treatment of IA remain challenging. Given the broad range of non-specific clinical symptoms and the shortcomings of current diagnostic techniques, most patients are either diagnosed as “possible” or “probable” cases but not “proven”. Moreover, because of the lack of sensitive and specific tests, many high-risk patients receive an empirical therapy or a prolonged treatment of high-priced antifungal agents, leading to unnecessary adverse effects and a high risk of drug resistance. More precise diagnostic techniques alongside a targeted antifungal treatment are fundamental requirements for reducing the morbidity and mortality of IA. Monoclonal antibodies (mAbs) with high specificity in targeting the corresponding antigen(s) may have the potential to improve diagnostic tests and form the basis for novel IA treatments. This review summarizes the up-to-date application of mAb-based approaches in assisting IA diagnosis and therapy.     

抗KOD聚合酶单克隆抗体的制备

单克隆抗体具有高度的均一性和专一性。抗KOD聚合酶单克隆抗体,可以特异性结合于KOD聚合酶,抑制PCR反应前常温状态下的多聚酶活性,显著提高PCR反应的特异性。为了提高KOD聚合酶PCR反应的特异性,以KOD聚合酶为抗原,制备了两株抗KOD聚合酶的单克隆抗体。以大肠杆菌表达的重组蛋白KOD聚合酶为抗原免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与SP2/0骨髓瘤细胞融合,通过HAT选择培养、ELISA检测和有限稀释法克隆,建立了2株能稳定分泌抗KOD聚合酶单克隆抗体的杂交瘤细胞,分别命名为1F5与3G6,对应的抗体亚型分别是IgG1和IgG2a。染色体计数结果分别是85~90和83~86。1F5与3G6的细胞上清和腹水的ELISA效价分别为1∶28,1∶29和1∶211,1∶211。间接免疫荧光证实制备的单克隆抗体的特异性良好。该结果为进一步建立高特异性的PCR方法奠定了良好的基础。     

Detection of Myosin 1g Overexpression in Pediatric Leukemia by Novel Monoclonal Antibodies

Myosin 1g (Myo1g) is a mechanoenzyme associated with actin filaments, expressed exclusively in hematopoietic cells, and involved in various cellular functions, including cell migration, adhesion, and membrane trafficking. Despite the importance of Myo1g in distinct functions, there is currently no monoclonal antibody (mAb) against Myo1g. mAbs are helpful tools for the detection of specific antigens in tumor cells and other tissues. The development of mAbs against targeted dysregulated molecules in cancer cells remains a crucial tool for aiding in the diagnosis and the treatment of patients. Using hybridoma technology, we generated a panel of hybridomas specific for Myo1g. ELISA, immunofluorescence, and Western blot assay results revealed the recognition of Myo1g by these novel monoclonal antibodies in normal and transformed T and B cells. Here, we report the development and application of new monoclonal antibodies against Myo1g for their potential use to detect its overexpression in acute lymphoblastic leukemia (ALL) patients.     

Therapeutic Strategies of Biologics in Chronic Rhinosinusitis: Current Options and Future Targets

Chronic rhinosinusitis is a chronic inflammatory disease of the upper airways, for which treatment options include medical or surgical therapy. However, there are limitations to conservative treatment strategies, such as the relapse of nasal polyps. In this review, we discuss the rising role of biomolecular mechanisms associated with various biologics that have been approved or are undergoing clinical trials to treat chronic rhinosinusitis. We also highlight the potential molecular therapeutic targets for managing and treating chronic rhinosinusitis.     

Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.     

New Anti-Nodal Monoclonal Antibodies Targeting the Nodal Pre-Helix Loop Involved in Cripto-1 Binding

Nodal is a potent embryonic morphogen belonging to the TGF-β superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44–67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (K_D 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.     

Aqueous Two-Phase Extraction of Monoclonal Antibodies from High Cell Density Cell Culture

Improvements of the upstream process for the production of monoclonal antibodies (mAb) has resulted in mammalian cell cultures with significantly increased cell densities. These high cell density (HCD) broths pose severe challenges for established downstream unit operations due to the increased biomass load. To meet these challenges, aqueous two-phase extraction was examined as a potential method for the clarification, as well as first stage purification of a Chinese hamster ovary expressed mAb with cell densities up to 70 · 10⁶ cells mL⁻¹. A clarification with 99 % yield of mAb and 68 % DNA removal was accomplished, which contributes to enable a foundation to the industrial application of aqueous two-phase extraction for manufacturing of mAb from HCD cultivations.     

Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.     

肿瘤坏死因子α单克隆抗体和多克隆抗体的研制和鉴定

以rhTNF－α作为免疫原，通过杂交瘤技术建立了15株稳定分泌rhTNF－α单克隆抗体的细胞株，对其所分泌单抗的生物学特性进行了鉴定，并探索了其纯化工艺。制备了高纯度兔抗rbTNF－α多克隆抗体，其中和细胞毒能力达到或超过SIGMA公司同类产品水平。该抗体对LPS所致的免发热有明显的抑制作用。利用单抗和多抗建立了ELISA双抗体夹心法，敏感性高，稳定性好，特异性强，适宜于临床检测大量样品。     

Her-2 Targeted Therapy in Advanced Urothelial Cancer: From Monoclonal Antibodies to Antibody-Drug Conjugates

Metastatic urothelial cancer, associated with a poor prognosis, is still major cause of cancer-related death, with scarce options of effective treatment after progression to platinum-based chemotherapy and immunotherapy. The human epithelial growth factor receptor 2 (Her-2) has been identified as a new therapeutic target in medical oncology. However, despite the encouraging results in breast and gastric cancers, clinical trials with anti-Her-2 monoclonal antibodies and tyrosine-kinase inhibitors have shown limited efficacy of this strategy in urothelial tumors. Notably, more favorable data have been recently shown that antibody-drug conjugates are currently emerging as a novel promising approach for Her-2 targeted therapy in advanced urothelial cancer.     

治疗性狂犬病病毒单克隆抗体的研究进展

狂犬病是一种重要的人兽共患传染病,每年全世界约有上百万人暴露于该病。狂犬病严重暴露者应在接种狂犬病疫苗进行主动免疫的同时,接种马抗狂犬病病毒免疫球蛋白(ERIG)或人抗狂犬病病毒免疫球蛋白(HRIG)进行被动免疫。然而,应用ERIG或HRIG存在引起过敏反应或传播血液性疾病的危险,应用治疗性单克隆抗体可以克服上述缺点。本文就鼠源性狂犬病病毒单克隆抗体和人源化狂犬病病毒单克隆抗体的研究进展、作用机制及大规模研制等作一综述。     

CCL4 Regulates Eosinophil Activation in Eosinophilic Airway Inflammation

Eosinophilic chronic rhinosinusitis (ECRS) is a refractory airway disease accompanied by eosinophilic inflammation, the mechanisms of which are unknown. We recently found that CCL4/MIP-1β—a specific ligand for CCR5 receptors—was implicated in eosinophil recruitment into the inflammatory site and was substantially released from activated eosinophils. Moreover, it was found in nasal polyps from patients with ECRS, primarily in epithelial cells. In the present study, the role of epithelial cell-derived CCL4 in eosinophil activation was investigated. First, CCL4 expression in nasal polyps from patients with ECRS as well as its role of CCL4 in eosinophilic airway inflammation were investigated in an in vivo model. Furthermore, the role of CCL4 in CD69 expression—a marker of activated eosinophils—as well as the signaling pathways involved in CCL4-mediated eosinophil activation were investigated. Notably, CCL4 expression, but not CCL5, CCL11, or CCL26, was found to be significantly increased in nasal polyps from patients with ECRS associated with eosinophil infiltration as well as in BEAS-2B cells co-incubated with eosinophils. In an OVA-induced allergic mouse model, CCL4 increased eosinophil accumulation in the nasal mucosa and the bronchoalveolar lavage (BALF). Moreover, we found that CD69 expression was upregulated in CCL4-stimulated eosinophils; similarly, phosphorylation of several kinases, including platelet-derived growth factor receptor (PDGFR)β, SRC kinase family (Lck, Src, and Yes), and extracellular signal-regulated kinase (ERK), was upregulated. Further, CCR5, PDGFRβ, and/or Src kinase inhibition partially restored CCL4-induced CD69 upregulation. Thus, CCL4, which is derived from airway epithelial cells, plays a role in the accumulation and activation of eosinophils at inflammatory sites. These findings may provide a novel therapeutic target for eosinophilic airway inflammation, such as ECRS.     

Monoclonal Antibody Engineering and Design to Modulate FcRn Activities: A Comprehensive Review

Understanding the biological mechanisms underlying the pH-dependent nature of FcRn binding, as well as the various factors influencing the affinity to FcRn, was concurrent with the arrival of the first recombinant IgG monoclonal antibodies (mAbs) and IgG Fc-fusion proteins in clinical practice. IgG Fc–FcRn became a central subject of interest for the development of these drugs for the comfort of patients and good clinical responses. In this review, we describe (i) mAb mutations close to and outside the FcRn binding site, increasing the affinity for FcRn at acidic pH and leading to enhanced mAb half-life and biodistribution, and (ii) mAb mutations increasing the affinity for FcRn at acidic and neutral pH, blocking FcRn binding and resulting, in vivo, in endogenous IgG degradation. Mutations modifying FcRn binding are discussed in association with pH-dependent modulation of antigen binding and (iii) anti-FcRn mAbs, two of the latest innovations in anti-FcRn mAbs leading to endogenous IgG depletion. We discuss the pharmacological effects, the biological consequences, and advantages of targeting IgG–FcRn interactions and their application in human therapeutics.     

The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies

Since the approval of the first monoclonal antibody (mAb) in 1986, a huge effort has been made to guarantee safety and efficacy of therapeutic mAbs. As of July 2021, 118 mAbs are approved for the European market for a broad range of clinical indications. In order to ensure clinical efficacy and safety aspects, (pre-)clinical experimental approaches evaluate the respective modes of action (MoA). In addition to antigen-specificity including binding affinity and -avidity, MoA comprise Fc-mediated effector functions such as antibody dependent cellular cytotoxicity (ADCC) and the closely related antibody dependent cellular phagocytosis (ADCP). For this reason, a variety of cell-based assays have been established investigating effector functions of therapeutic mAbs with different effector/target-cell combinations and several readouts including Fcγ receptor (FcγR)-mediated lysis, fluorescence, or luminescence. Optimized FcγR-mediated effector functions regarding clinical safety and efficacy are addressed with modification strategies such as point mutations, altered glycosylation patterns, combination of different Fc subclasses (cross isotypes), and Fc-truncation of the mAb. These strategies opened the field for a next generation of therapeutic mAbs. In conclusion, it is of major importance to consider FcγR-mediated effector functions for the efficacy of therapeutic mAbs.     

CD37 in B Cell Derived Tumors—More than Just a Docking Point for Monoclonal Antibodies

CD37 is a tetraspanin expressed prominently on the surface of B cells. It is an attractive molecular target exploited in the immunotherapy of B cell-derived lymphomas and leukemia. Currently, several monoclonal antibodies targeting CD37 as well as chimeric antigen receptor-based immunotherapies are being developed and investigated in clinical trials. Given the unique role of CD37 in the biology of B cells, it seems that CD37 constitutes more than a docking point for monoclonal antibodies, and targeting this molecule may provide additional benefit to relapsed or refractory patients. In this review, we aimed to provide an extensive overview of the function of CD37 in B cell malignancies, providing a comprehensive view of recent therapeutic advances targeting CD37 and delineating future perspectives.     

小鼠单克隆抗体IgM Fabμ片段的制备

本文介绍了小鼠单克隆抗体IgM小分子活性片段的制备方法。在37℃和4℃下用胰蛋白酶消化IgM 4和24小时,其小片段用碘代乙酰胺烷基化,用Sephacryl S-200柱层析纯化。SDS-聚丙烯酰胺凝胶电泳证实:在37℃用胰酶裂解IgM,小片段分子量近50000(Fab),但其免疫活性较低:在4℃胰酶裂解IgM,小片段分子量为80 000～110 000(Fabμ),免疫活性与原IgM基本一致。我们用Fab μ片段与辣根过氧化物酶连接,其结合物用于双抗体夹心酶联免疫吸附试验,取得了满意的结果。     

Inhibition of Enzyme Activity of Rhipicephalus (Boophilus) microplus Triosephosphate Isomerase and BME26 Cell Growth by Monoclonal Antibodies

In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.     

Anti-PTK7 Monoclonal Antibodies Exhibit Anti-Tumor Activity at the Cellular Level and in Mouse Xenograft Models of Esophageal Squamous Cell Carcinoma

PTK7 is a catalytically defective receptor protein tyrosine kinase upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). In previous studies, we observed a positive correlation between PTK7 expression levels and tumorigenicity in various ESCC cell lines and xenograft mice with ESCC KYSE-30 cells. In this study, we analyzed the effects of anti-PTK7 monoclonal antibodies (mAbs) on the tumorigenic activity in KYSE-30 cells and in mouse xenograft models. PTK7 mAb-32 and mAb-43 bind with a high affinity to the extracellular domain of PTK7. PTK7 mAbs significantly reduced three-dimensional cell proliferation, adhesion, wound healing, and migration. PTK7 mAbs also reduce chemotactic invasiveness by decreasing MMP-9 secretion. PTK7 mAbs decreased actin cytoskeleton levels in the cortical region of KYSE-30 cells. PTK7 mAbs reduced the phosphorylation of ERK, SRC, and FAK. In a mouse xenograft model of ESCC using KYSE-30 cells, PTK7 mAbs reduced tumor growth in terms of volume, weight, and the number of Ki-67-positive cells. These results demonstrated that PTK7 mAbs can inhibit the tumorigenicity of ESCC at the cellular level and in vivo by blocking the function of PTK7. Considering the anticancer activities of PTK7 mAbs, we propose that PTK7 mAbs can be used in an effective treatment strategy for PTK7-positive malignancies, such as ESCC.