The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen

A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-Ig™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the flexibility of parental mAbs is important for designing and retaining functionality of DVD-Ig™ molecules. The architecture and dynamics of a DVD-Ig™ molecule and its parental mAbs was examined using single particle electron microscopy. Hinge angles measured for the DVD-Ig™ molecule were similar to the inner antigen parental mAb. The outer binding domain of the DVD-Ig™ molecule was highly mobile and three-dimensional (3D) analysis showed binding of inner antigen caused the outer domain to fold out of the plane with a major morphological change. Docking high-resolution X-ray structures into the 3D electron microscopy map supports the extraordinary domain flexibility observed in the DVD-Ig™ molecule allowing antigen binding with minimal steric hindrance.     

Production of immunoglobulin A in different reactor configurations

A murine hybridoma line (Zac3), secreting an IgA monoclonal antibody, was cultivated in different systems: a BALB/c mouse, a T-flask, a stirred-tank bioreactor and a hollow fiber reactor. These systems were characterized in terms of cell metabolism and performances for IgA production. Cultures in T-flask and batch bioreactor were found to be glutamine-limited. Ammonia and lactate were produced in significant amounts. IgA productivity was found to be constant and growth associated. Final IgA concentration was similar in both systems. In fed-batch cultures, supplemented with glutamine and glucose, maximum viable cell concentration was increased by 60% and final IgA concentration by 155%. The hollow fiber reactor was able to produce very large amounts of IgA at very high concentrations, similar to the value found in ascites fluid. The productivity ofZac3 is similar to the values reported for IgG-producing cell lines.     

Hybridomas: A new dimension in biological analyses

Summary Since the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic increase in publications using these reagents has been in itself fascinating and informative. An overview of the development of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules, differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the regulation of growth control and cell division are provided. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association. Washington, D.C., June 7–11. 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

Structure of the human secretory immunoglobulin M core

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A hyman hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by Pseudomonas aeruginosa exotoxin A

By fusing a human hybridoma producing an IgG2 antibody against human A431 epidermoid carcinoma cells with an Epstein-Barr virus-transformed human B lymphocyte producing an IgG2 antibody against Pseudomonas aeruginosa exotoxin A, we established a hybrid hybridoma producing a bispecific monoclonal antibody reacting with both A431 cells and the exotoxin. Human IgG was purified from the culture supernatant of the hybrid hybridoma, and the bispecific monoclonal antibody in the IgG preparation was further separated from the two parental antibodies by hydroxyapatite high-performance liquid chromatography. The human bispecific monoclonal antibody thus obtained efficiently targeted the antibody-reative cells, A431, for attack by the exotoxin in vitro.Abbreviations bs mAb Bispecific Monoclonal Antibody - HRP Horseradish Peroxidase - MHA Mixed Hemadsorption Assay - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide - PEA Pseudomonas aeruginosa Exotoxin A - PEG Polyethylene Glycol     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.     

基因工程双特异抗体研究进展

双特异抗体特别是双特异性单链抗体、亮氨酸接链双特异性抗体、双特异性单链抗体毒素等是近几年来发展起来的很有前途的监床诊疗生物制剂 ,本文对其基因构建、边接肽设计、表达产物及活性等方面的新进展作了重点论述。     

The making of bispecific antibodies

During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The ‘zoo’ of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term ‘developability’. In addition, different ‘target product profiles’, i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not ‘one best format’ for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

Lysozyme stimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes

Lysozyme [EC 3.2.1.17] derived from hen egg white stimulated immunoglobulin production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by the addition of lysozyme at 380 μg/ml in a serum-free medium. The immunoglobulin production stimulating effect of lysozyme was observed immediately after inoculation and maintained for 5 days. Lysozyme enhanced immunoglobulin production by the hybridoma line without growth promotion. This enzyme also accelerated IgM and IgG production of human peripheral blood lymphocytes 5.3-fold and 2.3-fold, respectively. These results suggest that lysozyme stimulates immunoglobuling production of not only specific hybridoma line, but also non-specific immunoglobulin producers. However, although the enzymatic activity of lysozyme was almost lost by heat-treatment at 100 °C for 30 min, the IPSF activity was retained. This fact suggests that IPSF activity of lysozyme does not come from its enzymatic activity or reaction products. All these findings clearly indicate that lysozyme has a novel function as an immunoglobulin production stimulating factor. GAPDH - glyceraldehyde-3-phosphate dehydrogenase; Ig - immunoglobulin; IPSF - immunoglobulin production stimulating factor; PBL - peripheral blood lymphocytes; HPLC - high-performance liquid chromatography. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase

Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 μg/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400–7000 bp slightly stimulated IPSF activity at 0.06 μg/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell. This revised version was published online in August 2006 with corrections to the Cover Date.     

Species specificity of hybridoma antibodies to surface antigens of guinea pig sperm

The species specificity of hybridoma antibodies to sperm surface antigens was studied. A collection of over 50 hybridoma antibodies that bind to the guinea pig sperm surface was tested for binding to mouse, rat, hamster, and human sperm by indirect immunofluorescence. None of the antibodies bind to mouse sperm. rat sperm, or human sperm. All but three of the antibodies also fail to bind to hamster sperm. AH-30, AH-31, and AH-1032, the three antibodies that crossreact with hamster sperm, show a different topographical localization on hamster sperm from that seen on guinea pig sperm. The three antibodies do not precipitate a ¹²⁵I surface-labeled antigen from hamster sperm extracts. However, from guinea pig sperm extracts, all three antibodies precipitate ¹²⁵I surface-labeled polypeptides with molecular weights (M_r) of 62,000, 52,000, and 38,000. This result suggests that the crossreacting antibodies may be recognizing different antigens on hamster and guinea pig sperm.     

7th Annual European Antibody Congress 2011

The 7th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The 2011 version of the EAC was attended by nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The first day focused on advances in understanding structure-function relationships, choosing the best format, glycoengineering biobetter antibodies, improving the efficacy and drugability of mAbs and epitope mapping. On the second day, the discovery of novel targets for mAb therapy, clinical pipeline updates, use of antibody combinations to address resistance, generation and identification of mAbs against new targets and biosimilar mAb development were discussed. Antibody-drug conjugates, domain antibodies and new scaffolds and bispecific antibodies were the topics of the third day. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors.     

8th Annual European Antibody Congress 2012

The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics.

The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors.     

Fabs-in-tandem immunoglobulin is a novel and versatile bispecific design for engaging multiple therapeutic targets

In recent years, the development of bispecific antibody (bsAb) has become a major trend in the biopharmaceutical industry. By simultaneously engaging 2 molcular targets, bsAbs show unique mechanisms of action that could lead to clinical benefits unattainable by conventional monoclonal antibodies. Various bsAb generation formats have been described, and several are being investigated in clinical development. However, some bsAb constructs have proven to be problematic due to their unfavorable physicochemical and pharmacokinetic properties, as well as poor manufacturing efficiencies. We describe here a new bispecific design, Fabs-in-tandem immunoglobulin (FIT-Ig), in which 2 antigen-binding fragments are fused directly in a crisscross orientation without any mutations or use of peptide linkers. This unique design provides a symmetric IgG-like bispecific molecule with correct association of 2 sets of VH/VL pairs. We show that FIT-Ig molecules exhibit favorable drug-like properties, in vitro and in vivo functions, as well as manufacturing efficiency for commercial development.     

双特异抗体的研究进展

双特异抗体是指可以同时结合两个不同抗原或一个抗原不同表位的特殊抗体,目前已有3个双特异抗体批准上市,还有很多个双特异抗体处于临床或临床前研究阶段。文中就双特异抗体的发现、制备方法、结构类型和设计策略、作用机制以及目前研究现状进行综述。