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  • 机译 实验和计算观察结果提供的免疫球蛋白G Fc区的动态视图
    摘要:The Fc portion of immunoglobulin G (IgG) is a horseshoe-shaped homodimer, which interacts with various effector proteins, including Fcγ receptors (FcγRs). These interactions are critically dependent on the pair of N-glycans packed between the two CH2 domains. Fucosylation of these N-glycans negatively affects human IgG1-FcγRIIIa interaction. The IgG1-Fc crystal structures mostly exhibit asymmetric quaternary conformations with divergent orientations of CH2 with respect to CH3. We aimed to provide dynamic views of IgG1-Fc by performing long-timescale molecular dynamics (MD) simulations, which were experimentally validated by small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our simulation results indicated that the dynamic conformational ensembles of Fc encompass most of the previously reported crystal structures determined in both free and complex forms, although the major Fc conformers in solution exhibited almost symmetric, stouter quaternary structures, unlike the crystal structures. Furthermore, the MD simulations suggested that the N-glycans restrict the motional freedom of CH2 and endow quaternary-structure plasticity through multiple intramolecular interaction networks. Moreover, the fucosylation of these N-glycans restricts the conformational freedom of the proximal tyrosine residue of functional importance, thereby precluding its interaction with FcγRIIIa. The dynamic views of Fc will provide opportunities to control the IgG interactions for developing therapeutic antibodies.
  • 机译 基于膜的水两相系统分离对单克隆抗体的综合澄清和纯化
    摘要:Therapeutic monoclonal antibodies (mAb) are used for the treatment of numerous serious diseases, which have led to an increasing demand over the last decades. Increased cell density and mAb titer of the cultivation broth lead to great challenges for the subsequent clarification and capture operations in the downstream process. As an alternative approach to the conventional downstream process, a selective mAb extraction via an aqueous two-phase system (ATPS) directly from the cultivation broth of a mAb producing industrial relevant chinese hamster ovary (CHO) cell line was investigated. An efficient purification of the mAb was accomplished by the ATPS composition. The phase separation was realized by a newly developed membrane based phase separator. Moreover, a complete cell removal was integrated into this process by the used membrane. A selectivity between both phases was achieved by membrane modification. Yields up to 93% in the light phase and removal of process related impurities were obtained after aqueous two-phase extraction (ATPE). Phase separation performance as well as contact angles on the membrane were characterized for different ATPS. ATPE directly from the cultivation broth in combination with the new membrane based phase separation led to a mAb yield of 78% with a simultaneous reduction of deoxyribonucleic acid (DNA) and host cell protein (HCP) load.
  • 机译 通过人类合成Fab噬菌体展示文库淘选针对中东呼吸系统综合症冠状病毒的单克隆抗体并进行表征
    摘要:Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed. Here, we used phage display to develop novel monoclonal antibodies (mAbs) that target MERS-CoV. A human Fab phage display library was panned against the S2 subunit of the MERS-CoV spike protein (MERS-S2P), yielding three unique Fabs (S2A3, S2A6, and S2D5). The Fabs had moderate apparent affinities (Half maximal effective concentration (EC50 = 123–421 nM) for MERS-S2P, showed no cross-reactivity to spike proteins from other CoVs, and were non-aggregating and thermostable (Tm = 61.5–80.4 °C). Reformatting the Fabs into IgGs (Immunoglobulin Gs) greatly increased their apparent affinities (KD = 0.17–1.2 nM), presumably due to the effects of avidity. These apparent affinities were notably higher than that of a previously reported anti-MERS-CoV S2 reference mAb (KD = 8.7 nM). Furthermore, two of the three mAbs (S2A3 and S2D5) bound only MERS-CoV (Erasmus Medical Center (EMC)) and not other CoVs, reflecting their high binding specificity. However, the mAbs lacked MERS-CoV neutralizing activity. Given their high affinity, specificity, and desirable stabilities, we anticipate that these anti-MERS-CoV mAbs would be suitable reagents for developing antibody-based diagnostics in laboratory or hospital settings for point-of-care testing.
  • 机译 抗体转移的回胚过程(B2G)
    摘要:Bispecific antibodies (bsAbs) with avidity-enhanced specificity can be used to address target cells with increased specificity, ideally binding efficiently to cells that express two cognate antigens, yet not to cells that express only one of those. Building blocks required to generate such bsAbs are binders that recognize the two antigens with high specificity yet with various (including very low monovalent) affinities. The herein described ‘back-to-germline’ (B2G) procedure defines such derivatives. It converts parent antibodies with high specificity to derivatives that retain specificity but modulate affinity. The approach defines mutations to be introduced into antibody complementarity-determining regions (CDRs) regions without requiring structures of antibody-antigen complexes. Instead, it reverses the B-cell maturation process that increases affinities, with preference on CDR residues with high antigen contact probability. Placing germline residues at those positions generates VH and VL domains and Fv-combinations thereof that retain specificities but are ‘de-matured’ to different degrees. De-maturation influences on-rates and off-rates, and can produce entities with extremely low affinity for which binding can only be detected in bivalent formats. A comparison with alanine replacement in CDRs (so far, the most frequently applied technology) indicates that B2G may be more reliable/predictable without introduction of stickiness or poly-reactivity. The applicability for generating sets of affinity-modulated monospecific variants is exemplarily shown for antibodies that bind CD138, Her2/neu, and EGFR.
  • 机译 抗体轻链框架的优化可增强表达,生物物理特性和药代动力学
    摘要:Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody. The amendment of the antibody sequence was based on homology to human germline VL genes. Three regions or positions were identified in the VL domain—L1-4, L66, L79—and mutated independently or in combination to match the closest germline V gene. None of the mutations altered oxMIF specificity or affinity, but some variants improved thermal stability, aggregation propensity, and resulted in up to five-fold higher expression. Importantly, the improved biopharmaceutical properties translated into a superior pharmacokinetic profile of the antibody. Thus, optimization of the V domain framework can ameliorate the biophysical qualities of a therapeutic antibody candidate, and as result its manufacturability, and also has the potential to improve pharmacokinetics.
  • 机译 抗病毒和癌症基因组DNA突变酶APOBEC3B的兔单克隆抗体
    摘要:The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.
  • 机译 双特异性T细胞重定向与嵌合抗原受体(CAR)-T细胞作为杀死癌细胞的方法
    摘要:The concepts for T-cell redirecting bispecific antibodies (TRBAs) and chimeric antigen receptor (CAR)-T cells are both at least 30 years old but both platforms are just now coming into age. Two TRBAs and two CAR-T cell products have been approved by major regulatory agencies within the last ten years for the treatment of hematological cancers and an additional 53 TRBAs and 246 CAR cell constructs are in clinical trials today. Two major groups of TRBAs include small, short-half-life bispecific antibodies that include bispecific T-cell engagers (BiTE®s) which require continuous dosing and larger, mostly IgG-like bispecific antibodies with extended pharmacokinetics that can be dosed infrequently. Most CAR-T cells today are autologous, although significant strides are being made to develop off-the-shelf, allogeneic CAR-based products. CAR-Ts form a cytolytic synapse with target cells that is very different from the classical immune synapse both physically and mechanistically, whereas the TRBA-induced synapse is similar to the classic immune synapse. Both TRBAs and CAR-T cells are highly efficacious in clinical trials but both also present safety concerns, particularly with cytokine release syndrome and neurotoxicity. New formats and dosing paradigms for TRBAs and CAR-T cells are being developed in efforts to maximize efficacy and minimize toxicity, as well as to optimize use with both solid and hematologic tumors, both of which present significant challenges such as target heterogeneity and the immunosuppressive tumor microenvironment.
  • 机译 双特异性抗体的设计与生产
    摘要:With the current biotherapeutic market dominated by antibody molecules, bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike the conventional monoclonal antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, production, and quality of these molecules, which will enable this important class of biologics to reach their therapeutic potential.
  • 机译 用于抗体治疗发现和开发的噬菌体展示库
    摘要:Phage display technology has played a key role in the remarkable progress of discovering and optimizing antibodies for diverse applications, particularly antibody-based drugs. This technology was initially developed by George Smith in the mid-1980s and applied by John McCafferty and Gregory Winter to antibody engineering at the beginning of 1990s. Here, we compare nine phage display antibody libraries published in the last decade, which represent the state of the art in the discovery and development of therapeutic antibodies using phage display. We first discuss the quality of the libraries and the diverse types of antibody repertoires used as substrates to build the libraries, i.e., naïve, synthetic, and semisynthetic. Second, we review the performance of the libraries in terms of the number of positive clones per panning, hit rate, affinity, and developability of the selected antibodies. Finally, we highlight current opportunities and challenges pertaining to phage display platforms and related display technologies.
  • 机译 VHH-光敏剂结合用于过表达Met的肿瘤细胞的靶向光动力疗法
    摘要:Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.
  • 机译 类风湿关节炎患者,系统性红斑狼疮患者和健康对照之间基于EBNA1 IgM的区分
    摘要:Epstein–Barr Virus (EBV) has been associated with development of rheumatic connective tissue diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in genetically susceptible individuals. Diagnosis of RA and SLE relies on clinical criteria in combination with the presence of characteristic autoantibodies. In addition, antibodies to several EBV antigens have been shown to be elevated in patients with these diseases compared to healthy controls (HC). Here, we elaborated improved enzyme-linked immunosorbent assays for antibodies (IgM, IgA, IgG) to the EBV proteins Epstein-Barr Virus nuclear antigen (EBNA)1 and early antigen diffuse (EAD) in order to determine their potential diagnostic role. We showed that especially EBNA1 IgM distinguished RA from SLE and HCs and also distinguished SLE from HCs. EBNA1 IgA was almost as effective in differentiating RA from SLE and HC, while EAD IgG and IgA were able to discern SLE patients from RA patients and HCs. Collectively, these findings illustrate the potential diagnostic use of antibodies to EBV proteins to diagnose RA and to differentiate SLE from RA.
  • 机译 抗瓜氨酸化蛋白抗体在类风湿关节炎中的特异性
    摘要:Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. The majority of individuals with RA are positive for the disease-specific anti-citrullinated protein antibodies (ACPAs). These antibodies are primarily of cross-reactive nature, hence, the true autoantigen to ACPA remains unidentified. In this study, we analyzed the reactivity of RA sera to several post-translationally modified epitopes, in order to further characterize the specific nature of ACPAs by immunoassays. Substituting citrulline with other amino acids, e.g., D-citrulline, homo-citrulline and methyl-arginine illustrated that ACPAs are utmost specific for citrullinated targets. Collectively, these findings support that ACPAs and citrullinated targets are specific for RA, making citrulline-containing peptide targets the most effective assays for detection of ACPAs.
  • 机译 超强效双特异性骆驼VHH抗体通过gp41和gp120表位的组合中和HIV
    摘要:Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.
  • 机译 单域抗体作为治疗和成像剂,用于治疗中枢神经系统疾病
    摘要:Antibodies have become one of the most successful therapeutics for a number of oncology and inflammatory diseases. So far, central nervous system (CNS) indications have missed out on the antibody revolution, while they remain ‘hidden’ behind several hard to breach barriers. Among the various antibody modalities, single-domain antibodies (sdAbs) may hold the ‘key’ to unlocking the access of antibody therapies to CNS diseases. The unique structural features of sdAbs make them the smallest monomeric antibody fragments suitable for molecular targeting. These features are of particular importance when developing antibodies as modular building blocks for engineering CNS-targeting therapeutics and imaging agents. In this review, we first introduce the characteristic properties of sdAbs compared to traditional antibodies. We then present recent advances in the development of sdAbs as potential therapeutics across brain barriers, including their use for the delivery of biologics across the blood–brain and blood–cerebrospinal fluid (CSF) barriers, treatment of neurodegenerative diseases and molecular imaging of brain targets.
  • 机译 David vs. Goliath:抗体片段的结构,功能和临床前景
    摘要:Since the licensing of the first monoclonal antibody therapy in 1986, monoclonal antibodies have become the largest class of biopharmaceuticals with over 80 antibodies currently approved for a variety of disease indications. The development of smaller, antigen binding antibody fragments, derived from conventional antibodies or produced recombinantly, has been growing at a fast pace. Antibody fragments can be used on their own or linked to other molecules to generate numerous possibilities for bispecific, multi-specific, multimeric, or multifunctional molecules, and to achieve a variety of biological effects. They offer several advantages over full-length monoclonal antibodies, particularly a lower cost of goods, and because of their small size they can penetrate tissues, access challenging epitopes, and have potentially reduced immunogenicity. In this review, we will discuss the structure, production, and mechanism of action of EMA/FDA-approved fragments and of those in clinical and pre-clinical development. We will also discuss current topics of interest surrounding the potential use of antibody fragments for intracellular targeting and blood–brain barrier (BBB) penetration.
  • 机译 IMGT®和30年的免疫信息学洞察抗体V和C结构域和功能
    摘要:At the 10th Human Genome Mapping (HGM10) Workshop, in New Haven, for the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as ‘genes’, as were the conventional genes. Under these HGM auspices, IMGT®, the international ImMunoGeneTics information system®, was created in June 1989 at Montpellier (University of Montpellier and CNRS). The creation of IMGT® marked the birth of immunoinformatics, a new science, at the interface between immunogenetics and bioinformatics. The accuracy and the consistency between genes and alleles, sequences, and three-dimensional (3D) structures are based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts: IMGT standardized keywords (IDENTIFICATION), IMGT gene and allele nomenclature (CLASSIFICATION), IMGT standardized labels (DESCRIPTION), IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION). These concepts provide IMGT® immunoinformatics insights for antibody V and C domain structure and function, used for the standardized description in IMGT® web resources, databases and tools, immune repertoires analysis, single cell and/or high-throughput sequencing (HTS, NGS), antibody humanization, and antibody engineering in relation with effector properties.
  • 机译 人IgG的配体及其效应子功能
    摘要:Activation of the humoral immune system is initiated when antibodies recognize an antigen and trigger effector functions through the interaction with Fc engaging molecules. The most abundant immunoglobulin isotype in serum is Immunoglobulin G (IgG), which is involved in many humoral immune responses, strongly interacting with effector molecules. The IgG subclass, allotype, and glycosylation pattern, among other factors, determine the interaction strength of the IgG-Fc domain with these Fc engaging molecules, and thereby the potential strength of their effector potential. The molecules responsible for the effector phase include the classical IgG-Fc receptors (FcγR), the neonatal Fc-receptor (FcRn), the Tripartite motif-containing protein 21 (TRIM21), the first component of the classical complement cascade (C1), and possibly, the Fc-receptor-like receptors (FcRL4/5). Here we provide an overview of the interactions of IgG with effector molecules and discuss how natural variation on the antibody and effector molecule side shapes the biological activities of antibodies. The increasing knowledge on the Fc-mediated effector functions of antibodies drives the development of better therapeutic antibodies for cancer immunotherapy or treatment of autoimmune diseases.
  • 机译 自身抗体在系统性血管炎中的诊断和临床应用
    摘要:Considerable progress has been made in understanding the role of autoantibodies in systemic vasculitides (SV), and consequently testing for anti-neutrophil cytoplasmic antibodies (ANCA), anti-glomerular basement membrane antibodies (anti-GBM), and anti-C1q antibodies is helpful and necessary in the diagnosis, prognosis, and monitoring of small-vessel vasculitis. ANCA-directed proteinase 3 (PR3-) or myeloperoxidase (MPO-) are sensitive and specific serologic markers for ANCA-associated vasculitides (AAV), anti-GBM antibodies are highly specific for the patients with anti-GBM antibody disease (formerly Goodpasture’s syndrome), and autoantibodies to C1q are characteristic of hypocomlementemic urticarial vasculitis syndrome (HUVS; anti-C1q vasculitis). The results of a current EUVAS study have led to changes in the established strategy for the ANCA testing in small-vessel vasculitis. The revised 2017 international consensus recommendations for ANCA detection support the primary use PR3- and MPO-ANCA immunoassays without the categorical need for additional indirect immunofluorescence (IIF). Interestingly, the presence of PR3- and MPO-ANCA have led to the differentiation of distinct disease phenotype of AAV: PR3-ANCA-associated vasculitis (PR3-AAV), MPO-ANCA-associated vasculitis (MPO-AAV), and ANCA-negative vasculitis. Further studies on the role of these autoantibodies are required to better categorize and manage appropriately the patients with small-vessel vasculitis and to develop more targeted therapy.
  • 机译 单域抗体及其与病毒感染作斗争的形式
    摘要:Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as Nanobodies®, have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor VHHs over conventional antibody formats for many applications. The exceptional ease by which it is possible to fuse VHHs with different molecular modules has been particularly explored in the context of viral infections. In this review, we focus on VHH formats that have been developed to combat viruses including influenza viruses, human immunodeficiency virus-1 (HIV-1), and human respiratory syncytial virus (RSV). Such formats may significantly increase the affinity, half-life, breadth of protection of an antiviral VHH and reduce the risk of viral escape. In addition, VHHs can be equipped with effector functions, for example to guide components of the immune system with high precision to sites of viral infection.
  • 机译 通过使用类似TCR的抗体靶向MHC配体
    摘要:Monoclonal antibodies (mAbs) are valuable as research reagents, in diagnosis and in therapy. Their high specificity, the ease in production, favorable biophysical properties and the opportunity to engineer different properties make mAbs a versatile class of biologics. mAbs targeting peptide–major histocompatibility molecule (pMHC) complexes are often referred to as “TCR-like” mAbs, as pMHC complexes are generally recognized by T-cell receptors (TCRs). Presentation of self- and non-self-derived peptide fragments on MHC molecules and subsequent activation of T cells dictate immune responses in health and disease. This includes responses to infectious agents or cancer but also aberrant responses against harmless self-peptides in autoimmune diseases. The ability of TCR-like mAbs to target specific peptides presented on MHC allows for their use to study peptide presentation or for diagnosis and therapy. This extends the scope of conventional mAbs, which are generally limited to cell-surface or soluble antigens. Herein, we review the strategies used to generate TCR-like mAbs and provide a structural comparison with the analogous TCR in pMHC binding. We further discuss their applications as research tools and therapeutic reagents in preclinical models as well as challenges and limitations associated with their use.

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