Antibody-mediated delivery of T-cell epitopes to antigen-presenting cells induce strong CD4 and CD8 T-cell responses

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10–100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells

Complement Fixing Polysaccharides from Terminalia macroptera Root Bark, Stem Bark and Leaves

The root bark, stem bark and leaves of Terminalia macroptera were sequentially extracted with ethanol, 50% ethanol-water, and 50 °C and 100 °C water using an accelerated solvent extractor. Ten bioactive purified polysaccharide fractions were obtained from those crude extracts after anion exchange chromatography and gel filtration. The polysaccharides and their native extracts were characterized with respect to molecular weight, chemical compositions and effects in the complement assay. The chemical compositions showed that the polysaccharides are of pectic nature. The results indicated that there was no great difference of the complement fixation activities in the crude extracts from the different plant parts when extracting with the accelerated solvent extraction system. The purified polysaccharide fractions 100WTSBH-I-I and 100WTRBH-I-I isolated from the 100 °C water extracts of stem and root bark respectively, showed the highest complement fixation activities. These two fractions have rhamnogalacturonan type I backbone, but only 100WTSBH-I-I contains side chains of both arabinogalactan type I and II. Based on the yield and activities of the fractions studied those from the root bark gave highest results, followed by those from leaves and stem bark. But in total, all plant materials are good sources for fractions containing bioactive polysaccharides

Identification of a High Affinity Fc gamma RIIA-binding Peptide That Distinguishes Fc gamma RIIA from Fc gamma RIIB and Exploits Fc gamma RIIA-mediated Phagocytosis and Degradation

FcγRIIA is a key activating receptor linking immune complex formation with cellular effector functions. FcγRIIA has 93% identity with an inhibitory receptor, FcγRIIB, which negatively regulates FcγRIIA. FcγRIIA is important in the therapeutic action of several monoclonal antibodies. Binding molecules that discriminate FcγRIIA from FcγRIIB may optimize receptor activity and serve as a lead for development of therapeutics with FcγRIIA as a key target. Here we report the use of phage display libraries to select short peptides with distinct FcγRIIA binding properties. An 11-mer peptide (WAWVWLTETAV) was characterized that bound FcγRIIA with a Kd of 500 nm. It mediated cell internalization and degradation of a model antigen. The peptide-binding site on FcγRIIA was shown to involve Phe163 and the IgG binding amino acids Trp90 and Trp113. It is thus overlapping but not identical to that of IgG. Neither activating receptors FcγRI and FcγRIII, nor FcγRIIB, all of which lack Phe163, bound the peptide. This research was originally published in: Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology

Multivalent pIX phage display selects for distinct and improved antibody properties

Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration. This approach was combined with the use of an engineered helper phage known to improve antibody pIX display and retrieval. By direct comparison with pIII display, we demonstrate that antibody display using this pIX system translates into substantially improved retrieval of desired specificities with favorable biophysical properties in de novo selection. We show that the effect was due to less E. coli host toxicity during phage propagation conferred by the lack of a signal sequence. This pIX combinatorial display platform provides a generic alternative route for obtaining good binders with high stability and may thus find broad applicability