Browsing by Author "Dawson, Jeffrey"

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    An entirely cell-based system to generate single-chain antibodies against cell surface receptors.
    (2008) Chen, Yu-Hsun Jason
    The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen (Ag). Traditionally, the generation of single chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single chain Abs that does not require the use of purified protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high throughputs creening of arrayed phage clones, and characterization of recombinant single chain variable regions(scFvs). This strategy was used to generate a panel of single chain Abs specific for the innate immunity receptor Toll‐like receptor2 (TLR2). Once generated, individual scFvs were subcloned into an expression vector allowing the production of recombinant antibodies in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell‐based system efficiently generates Abs that bind native molecules displayed on cell surfaces, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. However, an inconvenience of this strategy is that it requires construction of a new library for each target TLR. This problem might be solved by using non‐immune antibody libraries to obtain antibodies against multiple TLRs. Non‐immune libraries contain a wide variety of antibodies but these are often low affinity, while immune libraries, derived from immunized animals, containa high frequency of high affinity antibodies, but are typically limited to a single antigen. In addition, it can be difficult to produce non‐immune libraries with sufficient complexity to select Abs against multiple Ags. Because the re‐assortment of VH and VL regions that occurs during antibody library construction greatly increases library complexity, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti‐hTLR2 antibody library for antibodies specific for other members of the TLR family. This procedure, which we refer to as homolog mining, proved to be effective. Using a cell‐based system to pan and screen our anti‐hTLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti‐murine TLR2, anti‐hTLR5, and anti‐hTLR6, bind specifically to their target, with no cross‐reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re‐assortment of VH and VL fragments during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes. In conclusion, we developed an entirely cell‐based method to generate antibodies that bind to native molecules on the cell surface, while eliminating the requirement of recombinant proteins and the risk of microbial component contamination. With homolog mining, this system is capable of generating antibodies not only against the original immunized Ag, but also against homologous Ags. In combination, this system proved to be an effective and efficient means for generating multiple antibodies that bind to multiple related Ags as they are displayed on cell surfaces.
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    Incorporation of CpG Oligodeoxynucleotides into α2-Macroglobulin: Development of a Novel Vaccine Adjuvant Delivery Mechanism
    (2007-05-02T14:53:43Z) Anderson, Ryan Berger
    Bacterial DNA is immunostimulatory, and the motifs responsible for this activity are unmethylated CpG dinucleotides. Following cellular uptake, CpG-containing oligodeoxynucleotides (CpG ODN) are trafficked to the endosome where they bind Toll-like receptor 9 (TLR9) to initiate a signaling cascade that culminates in the release of numerous pro-inflammatory cytokines. Because of their immunostimulatory properties, CpG ODN are being clinically evaluated as treatments and vaccine adjuvants for infectious diseases, cancer, and allergic disorders. α2-Macroglobulin (α2M) is a human plasma protein that binds and modulates the activity of a variety of cytokines, growth factors, enzymes, and antigens. Upon proteolytic activation, α2M is converted to its receptor recognized form, α2M*, and rapidly binds to and is internalized by immune competent cells expressing the α2M* endocytic receptor, LRP, and is then trafficked to the endosome. Based on these interactions, α2M seems to play an important role at sites of infection and inflammation by controlling the level of proteinase activity, modulating cytokine signals, and enhancing antigen processing for the adaptive immune response. Here, we report the first evidence that α2M* binds and forms stable complexes with nucleic acids. We have characterized the mechanisms and stoichiometry of this interaction, examined the pH and temperature stability of these complexes, and identified structural variables in the nucleic acids, namely length, base composition, and chemical modifications, that affect the nature of this interaction. We hypothesized that CpG ODN incorporation into α2M* may alter their immunostimulatory properties. Murine macrophages (MΦs) treated with α2M*-ODN complexes respond more rapidly and produce a greater cytokine response than those treated with free CpG ODN alone. Treating human PBMCs with α2M*-ODN complexes likewise demonstrated their enhanced ability to elicit immune responses. This was due to more rapid uptake and CpG ODN protection from degradation by extracellular nucleases. Co-incorporation of both protein ligands and CpG ODN into α2M* yields ternary complexes; these may permit the simultaneous delivery of both protein antigens and adjuvants to immune competent cells, potentially greatly enhancing the adaptive immune response and protective immunity. Based on the findings that incorporation into α2M* confers enhanced immunostimulatory activity of CpG ODN, this technology may be exploited to improve CpG ODN-based therapeutics by increasing efficacy, minimizing side effects, reducing dosing requirements, and reducing cost.