Dataset Information

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

ABSTRACT: Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

SUBMITTER: Sun Z

PROVIDER: S-EPMC9019674 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

Sun Zehua Z Li Wei W Mellors John W JW Orentas Rimas R Dimitrov Dimiter S DS

Frontiers in immunology 20220406

Phage display is a well-established technology for <i>in vitro</i> selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. ...[more]

PMID: 35464476

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Project description:Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.

Project description:Extensive diversity has been identified in the human heavy chain immunoglobulin locus, including allelic variation, gene duplication, and insertion/deletion events. Several genes have been suggested to be deleted in many haplotypes. Such findings have commonly been based on inference of the germline repertoire from data sets covering antibody heavy chain encoding transcripts. The inference process operates under conditions that may limit identification of genes transcribed at low levels. The presence of rare transcripts that would indicate the existence of poorly expressed alleles in haplotypes that otherwise appear to have deleted these genes has been assessed in the present study. Alleles IGHV1-2*05, IGHV1-3*02, IGHV4-4*01, and IGHV7-4-1*01 were all identified as being expressed from multiple haplotypes, but only at low levels, haplotypes that by inference often appeared not to express these genes at all. These genes are thus not as commonly deleted as previously thought. An assessment of the 5' untranslated region (up to and including the TATA-box), the signal peptide-encoding part of the gene, and the 3'-heptamer suggests that the alleles have no or minimal sequence difference in these regions in comparison to highly expressed alleles. This suggest that they may be able to participate in immunoglobulin gene rearrangement, transcription and translation. However, all four poorly expressed alleles harbor unusual sequence variants within their coding region that may compromise the functionality of the encoded products, thereby limiting their incorporation into the immunoglobulin repertoire. Transcripts based on IGHV7-4-1*01 that had undergone somatic hypermutation and class switch had mutated the codon that encoded the unusual residue in framework region 3 (cysteine 92; located far from the antigen binding site). This finding further supports the poor compatibility of this unusual residue in a fully functional protein product. Indications of a linkage disequilibrium were identified as IGHV1-2*05 and IGHV4-4*01 co-localized to the same haplotypes. Furthermore, transcripts of two of the poorly expressed alleles (IGHV1-3*02 and IGHV4-4*01) mostly do not encode in-frame, functional products, suggesting that these alleles might be essentially non-functional. It is proposed that the functionality status of immunoglobulin genes should also include assessment of their ability to encode functional protein products.

Dataset Information

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

Publications

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

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