Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.

Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific.

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.

A highly potent CD73 biparatopic antibody blocks organization of the enzyme active site through dual mechanisms.

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.

Quantitative Proteomic Profiling Reveals Novel Plasmodium falciparum Surface Antigens and Possible Vaccine Candidates.

Despite recent efforts toward control and elimination, malaria remains a major public health problem worldwide. Plasmodium falciparum resistance against artemisinin, used in front line combination drugs, is on the rise, and the only approved vaccine shows limited efficacy. Combinations of novel and tailored drug and vaccine interventions are required to maintain the momentum of the current malaria elimination program. Current evidence suggests that strain-transcendent protection against malaria infection can be achieved using whole organism vaccination or with a polyvalent vaccine covering multiple antigens or epitopes. These approaches have been successfully applied to the human-infective sporozoite stage. Both systemic and tissue-specific pathology during infection with the human malaria parasite P. falciparum is caused by asexual blood stages. Tissue tropism and vascular sequestration are the result of specific binding interactions between antigens on the parasite-infected red blood cell (pRBC) surface and endothelial receptors. The major surface antigen and parasite ligand binding to endothelial receptors, PfEMP1 is encoded by about 60 variants per genome and shows high sequence diversity across strains. Apart from PfEMP1 and three additional variant surface antigen families RIFIN, STEVOR, and SURFIN, systematic analysis of the infected red blood cell surface is lacking. Here we present the most comprehensive proteomic investigation of the parasitized red blood cell surface so far. Apart from the known variant surface antigens, we identified a set of putative single copy surface antigens with low sequence diversity, several of which are validated in a series of complementary experiments. Further functional and immunological investigation is underway to test these novel P. falciparum blood stage proteins as possible vaccine candidates.

Deubiquitinase FAM/USP9X interacts with the E3 ubiquitin ligase SMURF1 protein and protects it from ligase activity-dependent self-degradation.

Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration, and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ubiquitin ligase activity. This modular architecture allows for interactions with other proteins, which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is autoubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taken together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability.

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.