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      Design and production of novel tetravalent bispecific antibodies.

      Nature biotechnology

      Transfection, isolation & purification, chemistry, biosynthesis, Recombinant Fusion Proteins, Multiple Myeloma, Mice, Ligands, Kinetics, Immunoglobulin Variable Region, Immunoglobulin Heavy Chains, Immunoglobulin G, Immunoglobulin Constant Regions, Humans, Enzyme-Linked Immunosorbent Assay, Drug Design, Dansyl Compounds, Chromatography, Gel, Antibody Affinity, Antibodies, Bispecific, Animals

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          Abstract

          We have produced novel bispecific antibodies by fusing the DNA encoding a single chain antibody (ScFv) after the C terminus (CH3-ScFv) or after the hinge (Hinge-ScFv) with an antibody of a different specificity. The fusion protein is expressed by gene transfection in the context of a murine variable region. Transfectomas secrete a homogeneous population of the recombinant antibody with two different specificities, one at the N terminus (anti-dextran) and one at the C terminus (anti-dansyl). The CH3-ScFv antibody, which maintains the constant region of human IgG3, has some of the associated effector functions such as long half-life and Fc receptor binding. The Hinge-ScFv antibody which lacks the CH2 and CH3 domains has no known effector functions.

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          Most cited references 22

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          Site-directed mutagenesis by overlap extension using the polymerase chain reaction

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            The binding site for C1q on IgG.

            In humoral defence, pathogens are cleared by antibodies acting as adaptor molecules: they bind to antigen and trigger clearance mechanisms such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and complement lysis. The first step in the complement cascade is the binding of C1q to the antibody. There are six heads on C1q, connected by collagen-like stems to a central stalk, and the isolated heads bind to the Fc portion of antibody rather weakly, with an affinity of 100 microM (ref. 3). Binding of antibody to multiple epitopes on an antigenic surface, aggregates the antibody and this facilitates the binding of several C1q heads, leading to an enhanced affinity of about 10 nM (ref. 1). Within the Fc portion of the antibody, C1q binds to the CH2 domain. The interaction is sensitive to ionic strength, and appears to be highly conserved throughout evolution as C1q reacts with IgG from different species (for example see ref. 8). By systematically altering surface residues in the mouse IgG2b isotype, we have localized the binding site for C1q to three side chains, Glu 318, Lys 320 and Lys 322. These residues are relatively conserved in other antibody isotypes, and a peptide mimic of this sequence is able to inhibit complement lysis. We propose that this sequence motif forms a common core in the interactions of IgG and C1q.
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              Structural features of human immunoglobulin G that determine isotype- specific differences in complement activation

               MH Tao,  SL Morrison,  RI Smith (1993)
              Although very similar in sequence, the four subclasses of human immunoglobulin G (IgG) differ markedly in their ability to activate complement. Glu318-Lys320-Lys322 has been identified as a key binding motif for the first component of complement, C1q, and is present in all isotypes of Ig capable of activating complement. This motif, however, is present in all subclasses of human IgG, including those that show little (IgG2) or even no (IgG4) complement activity. Using point mutants of chimeric antibodies, we have identified specific residues responsible for the differing ability of the IgG subclasses to fix complement. In particular, we show that Ser at position 331 in gamma 4 is critical for determining the inability of that isotype to bind C1q and activate complement. Additionally, we provide further evidence that levels of C1q binding do not necessarily correlate with levels of complement activity, and that C1q binding alone is not sufficient for complement activation.
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                Author and article information

                Journal
                10.1038/nbt0297-159
                9035142

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