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Berlin Brandenburg

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  • 1
    Language: English
    In: The Journal of biological chemistry, 01 September 2017, Vol.292(35), pp.14706-14717
    Description: Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics.
    Keywords: Antibody ; Antibody Engineering ; Anticancer Drug ; Crystal Structure ; Immunoglobulin G (Igg) ; Models, Molecular ; Protein Engineering ; Antibodies, Bispecific -- Metabolism ; Antibodies, Monoclonal, Humanized -- Metabolism ; Antineoplastic Agents -- Metabolism ; Immunoglobulin G -- Metabolism ; Receptor, Erbb-2 -- Antagonists & Inhibitors ; Receptor, Erbb-3 -- Antagonists & Inhibitors
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 2
    Language: English
    In: Analytical Chemistry, August 21, 2012, Vol.84(16), p.7227(6)
    Description: Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.
    Keywords: Antibodies -- Research ; Gas Chromatography -- Usage ; Ion Implantation -- Methods ; Mass Spectrometry -- Usage
    ISSN: 0003-2700
    E-ISSN: 15206882
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  • 3
    Language: English
    In: Journal of Biological Chemistry, 03/29/1996, Vol.271(13), pp.7630-7634
    Description: This report describes the construction of leucine zip-per-based dimerization cassettes for the conversion of recombinant monomeric scFv antibody fragments to bivalent and bispecific dimers. A truncated murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned into four scFv fragments previously isolated from a synthetic antibody phage display library. Cysteine residues flanking the zipper region were introduced to covalently link dimerized scFv fragments. The secreted fusion proteins were shown to spontaneously and efficiently form stable Fos-Fos or Jun-Jun homodimers in the Escherichia coli periplasm at levels comparable to their monovalent counterparts. The bivalent (scFv) sub(2) fragments performed well in enzyme-linked immunosorbent assay, flowcytometric, and immunohistochemical analysis. Fos and Jun homodimer (scFv) sub(2) antibodies with different specificities could be reduced, reshuffled, and reoxidized to form preparations of functional bispecific (scFv) sub(2) Fos-Jun heterodimers. These Fos and Jun fusion protein cassettes provide a universal basis for the construction of dimeric scFv antibodies with enhanced avidity or dual specificity.
    Keywords: Escherichia Coli ; Escherichia Coli ; Leucine Zipper ; Antibodies ; Phage Display ; Gene Libraries ; Fusion Protein ; Leucine Zipper ; Antibodies ; Phage Display ; Gene Libraries ; Fusion Protein ; Transcription Factors ; Other ; Fv ; Fv ; Fv;
    ISSN: 0021-9258
    E-ISSN: 1083-351X
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  • 4
    In: Proceedings of the National Academy of Sciences of the United States, April 25, 1995, Vol.92(9), p.3938(5)
    Description: Antibody library and flow cytometry were combined to isolate phage antibodies which specifically bind to the antigens present on the surfaces of cells. The isolated phage antibodies bound mainly to the subpopulation used for selection and also bound to some other populations and cells in the mixture. Two phage antibodies displayed new staining patterns of B-lineage cells. Surface antigens of blood and fetal bone marrow cells are detected by this method which does not require immunization or the repeated formation of phage antibody libraries.
    Keywords: Monoclonal Antibodies -- Research
    ISSN: 0027-8424
    E-ISSN: 10916490
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  • 5
    Language: English
    In: Biotechnology and Bioengineering, 01 August 2010, Vol.106(5), pp.741-750
    Description: Therapeutic monoclonal antibodies, a highly successful class of biological drugs, are conventionally manufactured in mammalian cell lines. A recent approach to increase the therapeutic effectiveness of monoclonal antibodies has been to combine two or more of them; however this increases the complexity of development and manufacture. To address this issue a method to efficiently express multiple monoclonal antibodies from a single cell has been developed and we describe here the generation of stable cell clones that express high levels of a human monoclonal antibody mixture. PER.C6® cells were transfected with a combination of plasmids containing genes encoding three different antibodies. Clones that express the three corresponding antibody specificities were identified, subcloned, and passaged in the absence of antibiotic selection pressure. At several time points, batch production runs were analyzed for stable growth and IgG production characteristics. The majority (11/12) of subclones analyzed expressed all three antibody specificities in constant ratios with total IgG productivity ranging between 15 and 20 pg/cell/day under suboptimal culture conditions after up to 67 population doublings. The growth and IgG production characteristics of the stable clones reported here resemble those of single monoclonal antibody cell lines from conventional clone generation programs. We conclude that the methodology described here is applicable to the generation of stable PER.C6® clones for industrial scale production of mixtures of antibodies. Biotechnol. Bioeng. 2010;106: 741–750. © 2010 Wiley Periodicals, Inc.
    Keywords: Antibody Mixtures ; Per.C6® ; Oligoclonics™ ; Clonal Cell Lines
    ISSN: 0006-3592
    E-ISSN: 1097-0290
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  • 6
  • 7
    Language: English
    In: mAbs, 01 January 2014, Vol.6(1), pp.197-203
    Description: Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However,...
    Keywords: Native Mass Spectrometry ; Monoclonal Antibody ; Composite Mixtures ; Intact Protein Analysis ; High Resolution Mass Spectrometry ; Pharmacy, Therapeutics, & Pharmacology
    ISSN: 1942-0862
    E-ISSN: 1942-0870
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  • 8
    Language: English
    In: Future Virology, Sept, 2009, Vol.4(5), p.419(4)
    Description: Evaluation of: Sultana H, Foellmer HG, Neelakanta G et al.: Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and is recognized by a therapeutic cross-reactive human monoclonal antibody. J. Immunol. 183(1), 650-660 (2009). Flaviviruses, such as West Nile virus, the dengue serotypes, Japanese encephalitis virus and yellow fever virus are important human pathogens and can co-circulate in parts of the world. Humoral immunity is important in the mammalian protective response to flaviviruses and passive immunization has been shown to be effective in preventing infection and even treating disease. Monoclonal antibodies (mAbs) against flaviviruses can be divided basically into potent neutralizing mAbs with restricted flavivirus cross-reactivity and broadly cross-reactive mAbs with low neutralizing potency and protective activity. Here, Sultana and colleagues extend the characterization of the cross-reactive mAb11 by defining its epitope within the fusion loop of the West Nile virus envelope protein and link the reduced neuroinvasiveness of a mAb11 neutralization escape variant to a diminished ability to activate Toll-like receptor 3, induce inflammatory cytokine release and compromise blood-brain barrier integrity. The results highlight the importance of the fusion loop in viral pathogenesis; however, challenges remain to develop mAb11 into a viable therapeutic. Instead, mixtures of potent neutralizing mAbs with different flavivirus specificities may be a more feasible approach toward a broadly active flavivirus therapeutic antibody product.
    Keywords: Flavivirus Infections -- Development And Progression ; Flavivirus Infections -- Care And Treatment ; Flavivirus Infections -- Research ; Monoclonal Antibodies -- Physiological Aspects ; Monoclonal Antibodies -- Research ; Viral Envelopes -- Physiological Aspects ; Viral Envelopes -- Research
    ISSN: 1746-0794
    E-ISSN: 17460808
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  • 9
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 1997, Vol.94(8), pp.3903-3908
    Description: markdownabstractIn the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho–stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cellspecific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4–4, TB4–20, and TB4–28. While TB4–4 and TB4–20 are both epithelium specific, TB4–28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4–4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4–20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.
    Keywords: Sciences (General);
    ISSN: 00278424
    E-ISSN: 10916490
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  • 10
    Language: English
    In: Journal of Molecular Biology, 1995, Vol.248(1), pp.97-105
    Description: We have constructed a large (3.6x10 super(8) clones) phage display library of human single chain Fv (scFv) antibody fragments by combining 49 germline V sub(H) genes with synthetic heavy chain CDR3 (HCDR3) regions and seven light chains. The HCDR3 regions varied in length between 6 and 15 residues and were designed to contain fully randomized stretches of amino acid residues flanked by regions of limited residue variability that were composed of amino acid residues that frequently occur in natural antibodies. We reasoned that this approach would increase the frequency of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection on solid phase-bound antigens we obtained phage antibodies with binding activities to 13 different antigens, including Von Willebrand factor, the DNA-binding HMG box of transcription factor TCF-1 and the tumor antigen EGP-2. In addition, we applied a competitive selection procedure to target phage antibodies to the desired portion of a recombinant fusion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. The functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens, Western blotting assays and immunofluorescent analysis of cells by flow cytometry. The results demonstrate that this large human phage antibody library contains a broad assortment of binding specificities that can be applied in a variety of biochemical assays.
    Keywords: Phage Display ; Human Antibodies ; Synthetic Libraries ; Competitive Selection ; Designed Hcdr3 Regions ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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