Rapid On-Cell Selection of High-Performance Human Antibodies.
Ontology highlight
ABSTRACT:
SUBMITTER: Philpott DN
PROVIDER: S-EPMC8796304 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.
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Project description:Generating and improving antibodies and peptides that bind specifically to membrane protein targets such as ion channels and G protein-coupled receptors (GPCRs) can be challenging using established selection methods. Current strategies are often limited by difficulties in the presentation of the antigen or the efficiency of the selection process. Here, we report a method for obtaining antibodies specific for whole cell membrane-associated antigens which combines a cell-cell interaction format based on yeast display technology with fluorescence-activated cell sorting of dual fluorescent complexes. Using this method, we were able to direct the affinity maturation of an antagonist antibody specific for the proton-gated ion channel ASIC1a and showed that both the affinity and potency were improved. We were also able to use this method to do kinetic selections to generate clones with better dissociation profiles. In addition, this method was employed successfully to handle the difficult problem of selecting antibodies specific to a GPCR target, the mu-opioid receptor.
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Project description:Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC)-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.
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Project description:Post-translational modifications (PTMs) of proteins play critical roles in regulating many cellular events. Antibodies targeting site-specific PTMs are essential tools for detecting and enriching PTMs at sites of interest. However, fundamental difficulties in molecular recognition of both PTM and surrounding peptide sequence have hindered the efficient generation of highly specific anti-PTM antibodies. Furthermore, the widespread use of potentially inconsistent, non-renewable and molecularly undefined antibodies presents experimental challenges thought to contribute to the reproducibility crisis in biomedical research. In this study, we describe the structure-guided development of a platform that efficiently generates potent and selective recombinant antibodies to PTMs that are molecularly-defined and infinitely renewable. Our platform is built on our previous discovery of an unconventional binding mode of anti-PTM antibodies, antigen clasping, where two antigen binding sites cooperatively clasp a single antigen, creating extensive interactions with the antigen and leading to high selectivity and potency. We designed the platform that generates clasping antibodies with two distinct binding units, resulting in efficient generation of antibodies to a set of trimethylated histone H3 with high levels of specificity and affinity. Performance comparison in chromatin immunoprecipitation (ChIP), a common application in epigenomics, revealed that an anti-H3K27me3 clasping antibody exhibited superior specificity to a widely-used conventional antibody and captured symmetric and asymmetric nucleosomes in a less biased manner. We further generated clasping antibodies to phosphotyrosine antigens by using the same principle. These results suggest the broad applicability of our platform to generating high-performance clasping antibodies to diverse PTMs.
2024-12-02 | GSE270275 | GEO
Project description:In various clinical studies, Hodgkin's patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the nonhuman therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics.
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