Antibodies2014, 3(2), 192-204; doi:10.3390/antib3020192 - published online 8 April 2014 Show/Hide Abstract
Abstract: Immune responses directed against clotting factor FVIII (FVIII) seriously complicate treatments for patients with hemophilia A. This response can manifest in congenital hemophilia A patients who generate inhibitor antibodies that bind and inactivate “transplanted” replacement FVIII, as well as in acquired hemophiliacs, whose immune systems have lost tolerance to self-FVIII. Regardless of the mechanism by which production of anti-FVIII inhibitor antibody is triggered, the maintenance of this deleterious response in both congenital and acquired hemophiliacs likely relies upon FVIII specific memory B cells. In this review, the similarities and differences in the kinetics, specificities, and subclasses of antibodies produced in response to allo- and auto-FVIII is outlined. A brief description of the immune cell interactions that contribute to maintenance of antibody response, focusing on development of memory B cells and/or long lived plasma cells is also presented. As current treatments for inhibitor antibodies are not successful in all patients, a better understanding of the functions and persistence of memory B cells specific for FVIII is required. Herein, both clinical and experimental data regarding the effects of immune tolerance induction on memory B cell subpopulations is discussed. Finally, the outcomes of B cell-specific depletion via rituximab in hemophilia and other autoimmune diseases are discussed to highlight insights into the subpopulations of memory B cells that contribute to the development and maintenance of successful tolerance to FVIII.
Antibodies2014, 3(2), 182-191; doi:10.3390/antib3020182 - published online 2 April 2014 Show/Hide Abstract
Abstract: We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag) is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique.
Antibodies2014, 3(1), 169-181; doi:10.3390/antib3010169 - published online 21 March 2014 Show/Hide Abstract
Abstract: Anti-ferritin autoantibodies are found in many animals. Human ferritin-binding proteins (FBPs) were partially purified from human serum by ion-exchange chromatography and immobilized metal affinity chromatography with Zn2+. Crude FBPs were immunocoprecipitated with canine liver ferritin followed by the addition of anti-ferritin antibodies. Immunoglobulins in the immunocoprecipitate were detected with antibodies specific for human IgG, IgM or IgA heavy chains, and immunoglobulins IgG, IgM and IgA to bind to expressed recombinant human H and L chain homopolymers were also found. A portion of human serum proteins bound to zinc ions immobilized on beads were released upon the addition of canine liver ferritin, and the released protein was identified as IgM antibody. Additionally, the released proteins recognized peptide sequence (DPHLCDF) commonly found in amino acid sequences of mammalian ferritin H and L subunits. These results suggest that human serum contains anti-ferritin autoantibodies (IgG, IgM and IgA) which bind zinc ions and preferentially bind ferritin over both the H and L subunits, and that a portion of, but not all, the IgM antibodies bound to ferritin with higher affinity than to zinc ions and may recognize the common sequence found in mammalian ferritin H and L subunits.
Antibodies2014, 3(1), 155-168; doi:10.3390/antib3010155 - published online 12 March 2014 Show/Hide Abstract
Abstract: The production of human single-chain variable-fragments (scFvs) against carbohydrate antigens by phage display technology is seemingly a logical strategy towards the development of antibody therapeutics, since carbohydrates are self-antigens. Panning and screening of phages displaying human scFvs using a variety of neoglycolipids presenting structurally-defined carbohydrates resulted in a number of candidate phage clones as judged by cautious evaluation of DNA sequences and specific binding to carbohydrate moieties of interest. ScFv proteins were expressed in prokaryotic or eukaryotic cells from the respective genes. The characterization of isolated scFvs gene products after establishing expression, production and purification of scFv protein in different expression systems demonstrated that the production of scFv-human IgG1 Fc conjugates were originally sufficient in the media of stably-transfected cells, but declined during early passages. Bacterial expression of soluble scFv proteins with binding activity suffered low yields, whereas overexpressed scFv proteins formed inclusion bodies, which required refolding. An insect cell expression system producing soluble and active scFv proteins was found to be cost- and time-effective. The best expression system and fine adjustments for the conditions to prepare active forms had to be determined for each scFv protein. The successful production of active scFv proteins seems to be dependent on their DNA and/or amino acid sequences.
Antibodies2014, 3(1), 130-152; doi:10.3390/antib3010130 - published online 20 February 2014 Show/Hide Abstract
Abstract: Alloimmunization is an undesirable iatrogenic effect of transfusion and transplantation. In fact, recipients can be considered as responders or not responders, in a continuum from tolerance, including organ transplantation and transfusion, to polyimmunized and refractory patients. New models and large studies have enabled a better understanding of the mechanisms that induce specific alloantibody (alloAb) generation. Here, we focus on risk factors of alloimmunization. We review the alloantibody characteristics, summarize the different leukocytes involved in their induction, and suggest some hypotheses.