A fully automated high-throughput plasmid purification workstation for the generation of mammalian cell expression-quality DNA.

Early-stage antibody discovery and engineering typically require the cloning, expression, and screening of large numbers of proteins. Normally, DNA fragments encoding proteins of interest are cloned into extra-chromosomal plasmids that are amplified in Escherichia coli. Following purification from the bacteria, the plasmids are introduced into appropriate cells, and the expressed recombinant proteins screened for desired binding or function in a high-throughput manner. Even in a 96-well plate format, plasmid purification from E. coli is typically a labor intensive and time-consuming process. To further accelerate our existing biotherapeutic discovery workflows we designed, qualified, and enabled a fully integrated high-throughput plasmid purification and quantification workstation which we have termed AMPS (Automated Miniprep Plasmid Station). Using components from a commercially available kit, AMPS can purify plasmid preparations from twenty 96-deep-well plates of E. coli cultures, measure DNA absorbance at 260 nm, calculate plasmid concentrations, and prepare 96-deep-well plates for mammalian expression in an operator-independent manner. Plasmid yields and concentrations are equivalent to those obtained off-line. Furthermore, the quality of the DNA purified on the AMPS is equivalent to that obtained off-line in terms of DNA topology, and absence of contaminating bacterial chromosomal DNA and RNA. Most importantly, plasmids purified on the AMPS provide similar antibody titers following transfection in CHO cells as plasmids purified off-line. The AMPS bridges high-throughput E. coli colony picking capabilities typically available in an automation lab with downstream CHO expression needs and will facilitate screening of large numbers of biotherapeutics in binding and cell assay screens.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

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

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.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cß), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Molecular aspects of rheumatoid arthritis: role of transcription factors.

Rheumatoid arthritis is a multifactorial disease characterized by chronic inflammation of the joints. Both genetic and environmental factors are involved in the pathogenesis leading to joint destruction and ultimately disability. In the inflamed RA joint the synovium is highly infiltrated by CD4+ T cells, B cells and macrophages, and the intimal lining becomes hyperplastic owing to the increased number of macrophage-like and fibroblast-like synoviocytes. This hyperplastic intimal synovial lining forms an aggressive front, called pannus, which invades cartilage and bone structures, leading to the destruction and compromised function of affected joints. This process is mediated by a number of cytokines (tumor necrosis factor-alpha, interleukin-1, interleukin-6, interleukin-17 interferon-gamma, etc.), chemokines (monocyte chemoattractant protein-1, monocyte chemoattractant protein-4 CCL18, etc.), cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, etc.) and matrix metalloproteinases. Expression of these molecules is controlled at the transcription level and activation of a limited number of transcription factors is involved in this process.

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display.

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.