This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG and murine IgG generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™.
View Article and Find Full Text PDFImplementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies.
View Article and Find Full Text PDFAlthough humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position.
View Article and Find Full Text PDFFully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10.
View Article and Find Full Text PDFWhile myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation.
View Article and Find Full Text PDFMalarial parasites are exquisitely susceptible to a number of microtubule inhibitors but most of these compounds also affect human microtubules. Herbicides of the dinitroaniline and phosphorothioamidate classes however affect some plant and protozoal cells but not mammalian ones. We have previously shown that these herbicides block schizogony in erythrocytic parasites of the most lethal human malaria, Plasmodium falciparum, disrupt their mitotic spindles, and bind selectively to parasite tubulin.
View Article and Find Full Text PDFWe have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3.
View Article and Find Full Text PDFHighly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide.
View Article and Find Full Text PDFProtein engineering techniques can facilitate the direct de-convolution of specific domains, regions, and particular amino acids that contribute to protein function. Many tools are available to aid this enterprise and herein we describe one such tool, a technique we term "Molecular Scanning" (MS). MS is analogous to previously described alanine scanning in that it samples potentially functional sequence space, but differs in that it uses Error-Prone polymerase chain reaction to randomly introduce all amino acids across the sequence space, as opposed to simply introducing alanine at each desired position.
View Article and Find Full Text PDFExamination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions.
View Article and Find Full Text PDFJ Mol Biol
May 2009
Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized.
View Article and Find Full Text PDFPhage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration.
View Article and Find Full Text PDFMicrotubules play important roles in cell division, motility and structural integrity of malarial parasites. Some microtubule inhibitors disrupt parasite development at very low concentrations, but most of them also kill mammalian cells. However, the dinitroaniline family of herbicides, which bind specifically to plant tubulin, have inhibitory activity on plant cells but are relatively non-toxic to human cells.
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