Searching for common ground.

The conserved coronavirus fusion peptide is a target of broadly neutralizing antibodies.

Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

Structural and Biochemical Characterization of Cysteinylation in Broadly Neutralizing Antibodies to HIV-1.

Antibodies with exceptional breadth and potency have been elicited in some individuals during natural HIV-1 infection. Elicitation and affinity maturation of broadly neutralizing antibodies (bnAbs) is therefore the central goal of HIV-1 vaccine development. The functional properties of bnAbs also make them attractive as immunotherapeutic agents, which has led to their production and optimization for passive immunotherapy. This process requires in vitro manufacturing and monitoring of any heterogeneous expression, especially when subpopulations of antibodies are produced with varying levels of biological activity. Post-translational modification (PTM) of antibodies can contribute to heterogeneity and is the focus of this study. Specifically, we have investigated cysteinylation in a bnAb lineage (PCDN family) targeting the N332-glycan supersite on the surface envelope glycoprotein (Env) of HIV-1. This PTM is defined by capping of unpaired cysteine residues with molecular cysteine. Through chromatography and mass spectrometry, we were able to characterize subpopulations of cysteinylated and non-cysteinylated antibodies when expressed in mammalian cells. The crystal structures of two PCDN antibodies represent the first structures of a cysteinylated antibody and reveal that the cysteinylation in this case is located in CDRH3. Biophysical studies indicate that cysteinylation of these HIV-1 antibodies does not interfere with antigen binding, which has been reported to occur in other cysteinylated antibodies. As such, these studies highlight the need for further investigation of cysteinylation in anti-HIV and other bnAbs.

VRC34-Antibody Lineage Development Reveals How a Required Rare Mutation Shapes the Maturation of a Broad HIV-Neutralizing Lineage.

Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

U3-1402, a Novel HER3-Targeting Antibody-Drug Conjugate, for the Treatment of Colorectal Cancer.

HER3 is overexpressed in several cancers, including colorectal cancer. Although therapies with anti-HER3 antibodies have been investigated, significant clinical benefits have not been reported. U3-1402 is a novel HER3-antibody-drug conjugate (ADC) composed of the HER3 antibody patritumab and a novel topoisomerase I inhibitor, DX-8951 derivative (DXd). The sensitivity of DXd was evaluated by a growth inhibition assay. The antitumor activity of U3-1402 was evaluated in a murine xenograft model in which its effects on cells, with a range of HER3 expression levels, were compared with those of patritumab alone, irinotecan, control-ADC, and saline. In the growth inhibition assay, all colorectal cancer cell lines were sensitive to DXd. In the tumor xenograft model, significant tumor regression with U3-1402 was observed both in the DiFi cell line (high HER3 expression; KRAS wild type) and in SW620 (high HER3 expression; KRAS mutation), but no treatment effect was observed in Colo320DM (low HER3 expression). Notably, SW620 tumor growth was significantly suppressed with U3-1402 compared with the saline-treated group (P < 0.001) and showed greater activity compared with the irinotecan group. By contrast, patritumab alone, control-ADC, and saline did not significantly differ in tumor growth inhibition. The antitumor activity of U3-1402 was dependent on HER3 expression level, but not on KRAS mutation status. These results support further investigation of development strategies for U3-1402 in patients with HER3-expressing colorectal cancer.