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RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells.

ABSTRACT: Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, low starting template concentrations, and the need for all the primers to function under the same PCR conditions. Here we demonstrate several advantages to incorporating RNase H2-dependent PCR (rh-PCR) into a high-throughput, antibody-discovery platform. Firstly, rh-PCR eliminated primer dimer synthesis to below detectable levels, thereby eliminating clones with a false positive antibody titer. Secondly, by increasing the specificity of PCR, the rh-PCR primers increased the recovery of cognate antibody variable regions from single B-cells, as well as downstream recombinant antibody titers. Finally, we demonstrate that rh-PCR primers provide a more homogeneous sample pool and greater sequence quality in a Next Generation Sequencing-based approach to obtaining DNA sequence information from large numbers of cloned antibody cognate pairs. Furthermore, the higher specificity of the rh-PCR primers allowed for a better match between native antibody germline sequences and the VL/VH fragments amplified from single B-cells.

SUBMITTER: Crissman J

PROVIDER: S-EPMC7643965 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells.

Crissman John J Lin Yuhao Y Separa Kevin K Duquette Madeleine M Cohen Michael M Velasquez Candyd C Cujec Thomas T

PloS one 20201105 11

Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, lo ...[more]

PMID: 33152031

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Project description:Ifitm3 (interferon-inducible transmembrane protein 3) was previously identified as an endosomal antiviral effector protein that blocks viral infection1-4. In analyses of gene expression data from patients with B-cell leukemia and lymphoma, we identified Ifitm3 as one of the strongest predictors of poor clinical outcome. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, engagement of the B-cell receptor (BCR) and PI3K-activation strongly induced expression of Ifitm3 and phosphorylation of its N-terminus at Y20, resulting in accumulation at the cell surface. In B-cell leukemia, oncogenic kinases induced phosphorylation at Y20, resulting in constitutive plasma membrane localization of Ifitm3. Ifitm3¯ / ¯ naïve B-cells developed at normal numbers, however, B-cell activation upon antigen encounter, germinal center formation and production of antigen-specific antibodies were compromised. Likewise, oncogenes that typically induce development of leukemia and lymphoma failed to transform Ifitm3¯ / ¯ B-cells. Conversely, a phosphomimetic mutation of Y20 induced oncogenic PI3K-signaling and initiated transformation of premalignant B-cells into overt leukemia. Functional experiments revealed a previously unrecognized function of Ifitm3 as PIP3-scaffold and central amplifier of PI3K signaling downstream of the BCR and adhesion receptors. PI3K signal-amplification depends on binding of Ifitm3 to PIP3 via two lysine residues (K83 and K104) in its conserved intracellular loop. In Ifitm3¯ / ¯ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors, which impaired BCR-signaling and cellular adhesion. We conclude that phosphorylation of IFITM3 upon Bcell antigen-encounter induces a dynamic switch from antiviral effector functions in endosomes to a PI3K-amplification loop at the cell surface. IFITM3-dependent amplification of PI3K-signaling downstream of the BCR and adhesion receptors is critical to enable rapid expansion of B-cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3- dependent signaling complexes and amplify PI3K-signaling for malignant transformation.

Project description:The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson's disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies.

Dataset Information

RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells.

Publications

RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells.

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