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

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.

Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2

The SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.

Poorly expressed alleles of several human immunoglobulin heavy chain variable (IGHV) genes are common in the human population

ABSTRACTExtensive 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 germline repertoire from data sets covering antibody heavy chain encoding transcripts. The inference process operate under conditions that may limit identification of genes transcribed at low levels. The presence of rare transcripts that would indicate the presence of poorly expressed alleles in haplotypes that otherwise appear to have deleted these genes has now been assessed. Alleles IGHV1-2*05, IGHV1-3*02, IGHV4-4*01, and IGHV7-4-1*01 were all identified as being expressed at very low levels from multiple haplotypes, haplotypes that by inference often appeared not to express these genes at all. These alleles harbor unusual sequence variants that may compromise the functionality of the encoded products. Transcripts of two of these alleles to a large degree do not encode a functional product, suggesting that these alleles might be non-functional. It is proposed that the functionality status of immunoglobulin genes should also include assessment of their ability to encode functional protein products.

UBR E3 ligases and the PDIA3 protease control degradation of unfolded antibody heavy chain by ERAD

Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.

Genetic Interaction Between the Non-homologous End Joining Factors during B and T Lymphocyte Development: In Vivo Mouse Models

The non-homologous end joining (NHEJ) DNA repair pathway is the main mechanism to repair DNA double strand breaks (DSBs) throughout the whole cell cycle. During NHEJ, core Ku70 and Ku80 subunits form the Ku heterodimer; Ku binds DSBs and promotes the recruitment of accessory factors (e.g., DNA-PKcs, PAXX, Mri) and downstream core factors (XLF, Lig4 and XRCC4). DSBs are induced during the V(D)J recombination in developing B and T lymphocytes to increase the repertoire of B and T cell receptors. Furthermore, DSBs are generated during the class switch recombination (CSR) in mature B lymphocytes, providing distinct effector functions of antibody heavy chain constant regions. The NHEJ is required for both V(D)J recombination and CSR. During the last decades, new NHEJ proteins have been reported, increasing the complexity of the molecular pathway. Multiple in vivo mouse models were generated and characterized to identify specific functions of NHEJ factors in the adaptive immune system. Here, we are summarizing available mouse models lacking one or several NHEJ factors, with a particular focus on early B cell development.