Broad neutralization of SARS-related viruses by human monoclonal antibodies

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines

Assessing the binding properties of the anti-PD-1 antibody landscape using label-free biosensors.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs

Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependent cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action

Inhibitor sensitivity of <i>PMT</i> knockouts.

<p>For dilution assays, yeast cells were grown overnight in YSD and then adjusted to an OD600 of 1.0 using YSD, diluted in 10-fold increments, and then 3 µl of each dilution were spotted onto solid YSD media with varying concentrations of PMTi-3. The picture of the plate without PMTi-3 was taken after 48 hours, and the picture of the plates containing PMTi-3 were taken after 96 hours of incubation at 25^°C. YGLY4140 is the parent strain of YGLY6001 and YGLY4329, YGLY733 is the parent strain of YGLY3773 and YGLY5968, and NRRL -Y11430 is the parent strain of YGLY35032.</p

Western blot analysis of an IgG1 expressed in the presence of PMTi-3.

<p>The <i>PMT</i> wild type strain YGLY4280 was grown in 96 well deep-well plates and induced in medium containing no PMTi-3 (C) or increasing concentrations of PMTi-3 (0.0187, 0.0375, 0.075, 0.15, 0.3, 0.625, 1.25, 2.5, 5, 10 µg/ml). After overnight induction, 7 µl of each supernatant was separated by polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes. IgG1 was detected using an anti-human IgG (heavy and light chain) antibody. Hyperglycosylated heavy-chain (indicated by an asterisk) is barely detectable at PMTi-3 concentrations of 0.15 µg/ml and higher, however strain growth and fitness (strain lysis) was still unaffected at PMTi-3 concentrations of up to 1.25 µg/ml. A representation of the PMTi-3 increase in concentration and the relative reduction in cell fitness are illustrated by <i>light</i> and <i>dark gray triangles</i> respectively.</p

<i>PMT</i> expression profile by microarray analysis.

<p>The relative levels of PMT mRNA levels were assessed by microarray analysis under glycerol (<i>shaded bars</i>) or methanol (<i>black bars</i>) growth conditions as described in the Materials and Methods Section. mRNA levels for <i>ACT1</i> and <i>GPD</i> have been added for reference.</p