Antibody heavy chain CDR3 length-dependent usage of human IGHJ4 and IGHJ6 germline genes

Abstract

Background Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. In recent years, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Methods Using VH3–23 as a model framework, we analyzed the naturally and productively rearranged CDR-H3 diversity in human immune repertoire. With homology modeling, we further built VH3–23-based structural models to understand the spatial paratope and its influencing parameters. Results We observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Such CDR-H3 length dependent usage of human germline genes was not impacted by the choices of VH in the V(D)J recombination, ethnic background and health conditions. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Conclusions We quantitatively determined the CDR-H3 length-dependent usage of human germline genes, which makes it possible to design synthetic diversity fully mimicking that of natural immune repertoire. Our observations shed light on the design of next generation synthetic diversity with improved probability of success.

Convergent Sequence Features of Antiviral B Cells

“…Unsurprising was the bias towards gene family VH3 within the IgG restricted dataset, as VH3 offers a repertoire of increased germline complexity not afforded by other VH segment families. Likewise, the preference towards IGHJ4 falls in line with previous findings and has been suggested to be the result of tyrosine desirability at the CDRH3 locus, by which IGHJ4 contributes two tyrosine residues [33,47]. However, the increased usage of VH5 among Paramyxoviridae, in comparison to the other antigen specificity categories, was striking considering VH5 is comprised of two gene segments, suggesting a potential selective pressure towards the use of VH5 genes in the repertoire against PIV-3 F, as has been implicated for a class of HIV-1 antibodies [48].…”

A Platform of ADAPTive Scaffolds: Development of CDR-H3 -Hairpin Mimics into Covalent Inhibitors of the PD1/PDL1 Immune Checkpoint.

“…[30,31] Following these studies, we became interested to developing -hairpin scaffolds that could withhold longer loops (>10-residue long) while closely mimicking the native fold found in antibodies. In comparison to all CDRs, H3 loops are known to possess the largest variability in sequence [32,33] and length (4 up to >21 residues) [34] which drastically increases the span of conformational space generated [35,36] to maximize bounded states. [37][38][39][40] The vast majority of CDR-H3s possess a -bulge motif edging the loops [41,42] , yet the role played by this motif in the hairpin thermodynamic stability and scaffold rigidification remain largely unexplored.…”

Generation of synthetic antibody fragments with optimal complementarity determining region lengths for Notch-1 recognition

“…Previous studies have shown that NGS has four main applications in antibody phage display: library quality control, analysis of selection outputs, reconstruction of low-frequency clones, and rationale design based on natural repertoires ( Ravn et al, 2010 ; Zhang et al, 2011 ; Mahon et al, 2013 ; D'Angelo et al, 2014 ; Glanville et al, 2015 ; Yang et al, 2017 ; Jian et al, 2019 ; Valadon et al, 2019 ; Azevedo Reis Teixeira et al, 2021 ; Wang et al, 2021 ), which have resulted in some FDA approved antibodies ( Alfaleh et al, 2020 ). In this work, we used NGS information for identifying and reconstructing low-frequency clones with less-frequent CDRH3 lengths, for studying the pairing between CDRL3 and CDRH3 lengths in the selection output, and for fine-tuning the library length diversity.…”